Norovirus and sapovirus antigens

ABSTRACT

Immunogenic compositions that elicit immune responses against Norovirus and Sapovirus antigens are described. In particular, the invention relates to polynucleotides encoding one or more capsid proteins or other immunogenic viral polypeptides from one or more strains of Norovirus and/or Sapovirus, coexpression of such immunogenic viral polypeptides with adjuvants, and methods of using the polynucleotides in applications including immunization and production of immunogenic viral polypeptides and viral-like particles (VLPs). Methods for producing Norovirus- or Sapovirus-derived multiple epitope fusion antigens or polyproteins and immunogenic compositions comprising one or more immunogenic polypeptides, polynucleotides, VLPs, and/or adjuvants are also described. The immunogenic compositions of the invention may also contain antigens other than Norovirus or Sapovirus antigens, including antigens that can be used in immunization against pathogens that cause diarrheal diseases, such as antigens derived from rotavirus.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 11/603,913, filed Nov.22, 2006 now U.S. Pat. No. 7,527,801, which application claims thebenefit of U.S. provisional application No. 60/739,217, filed Nov. 22,2005, which applications are hereby incorporated by reference in theirentireties.

TECHNICAL FIELD

The present invention pertains generally to compositions that elicitimmune responses against Noroviruses and/or Sapoviruses. In particular,the invention relates to immunogenic compositions comprising nucleicacids encoding Norovirus and/or Sapovirus antigens, and/or immunogenicpolypeptides, including structural polypeptides, nonstructuralpolypeptides, and polyproteins, and fragments thereof, and/ormultiepitope fusion proteins, and/or viral-like particles derived fromone or more genotypes and/or isolates of Norovirus and Sapovirus.Immunogenic compositions, in addition may contain antigens other thanNorovirus or Sapovirus antigens, including antigens that can be used inimmunization against pathogens that cause diarrheal diseases, such asantigens derived from rotavirus. Methods of eliciting an immune responsewith the immunogenic compositions of the invention and methods oftreating a Norovirus and/or Sapovirus infection are also described.

BACKGROUND

Noroviruses (also known as Norwalk-like viruses or Norwalk viruses) andSapoviruses (also known as Sapporo-like viruses) are etiological agentsof acute gastroenteritis in adults and children (Green et al. J. Infect.Dis. 181 (Suppl 2):S322-330). Norviruses and Sapoviruses are members ofthe Caliciviridae family of small, nonenveloped viruses, 27-35 nm indiameter, containing a single-strand of positive-sense genomic RNA.Currently, Norviruses and Sapoviruses are the only two genera of theCaliciviridae family known to cause human disease.

Noroviruses cause greater than 90% of nonbacterial gastroenteritisoutbreaks and an estimated 23 million cases of gastroenteritis in theU.S. per year (Fankhauser et al. (2002) J. Infect. Dis. 186:1-7; MMWRMorb. Mortal Weekly Rep. (2000) 49:207-211). Although, the Norwalkstrain of Norovirus was the first discovered, it is now apparent thatthe Norwalk virus causes less than 10% of gastroenteritis cases, whereasother members of the Norovirus family, such as the Lordsdale virus,Toronto virus, and Snow Mountain virus, may cause 90% of cases(Fankhauser et al. (1998) J. Infect. Dis. 178:1571-1578; Nishida et al.(2003) Appl. Environ. Microbiol. 69(10):5782-6).

The symptoms of Norovirus infection include simultaneous diarrhea andvomiting as well as fever, headaches, chills and stomach-aches. Thecause of such symptoms may be related to the binding of Noroviruses tocarbohydrate receptors of intestinal epithelial cells, which results inan imbalance in ion transfer (Marionneau et al. (2002) Gastroenterology122:1967-1977; Hutson et al. (2003) J. Virol. 77:405-415). Extremelycontagious, Noroviruses can cause disease by infection with as few as 10virions. Although, otherwise healthy people infected with Norovirusesmay recover within 2-4 days, they may still shed virus for up to 2 weeksafter the onset of symptoms; hence, infected individuals should bequarantined for up to two weeks. Approximately 30-40% of infected peoplemay remain symptom-free, though spread infection by shedding of virus toothers who may be more susceptible to infection (Hutson et al. TrendsMicrobiol. 2004 June; 12(6):279-287).

In contrast, Sapoviruses are less prevalent in gastroenteritis outbreaksand infect mostly infants and children, though occasionally adults(Zintz et al. (2005) Infect. Genet. Evol. 5:281-290; Johansson et al.(2005) Scand. J. Infect. Dis. 37:200-204; Rockx et al. (2002) Clin.Infect. Dis. 35:246-253). Sapoviruses also cause diarrhea and vomitingand spread infection through viral shedding, which may last for up to 2weeks.

There remains a need for an improved therapy for treating patientshaving gastroenteritis associated with Norovirus or Sapovirus infectionand methods for preventing the spread of infection.

SUMMARY

The present invention provides immunogenic compositions comprisingNorovirus and Sapovirus antigens. In particular, the invention providespolynucleotides encoding one or more capsid proteins or fragmentsthereof and/or other immunogenic viral polypeptides or peptides from oneor more strains of Norovirus and/or Sapovirus.

Methods for producing Norovirus- or Sapovirus-derived multiple epitopefusion antigens or polyprotein fusion antigens are also described.Immunogenic polypeptides, peptides, and/or VLPs may be mixed orco-expressed with adjuvants (e.g., detoxified mutants of E. coliheat-labile toxins (LT) such as LT-K63 or LT-R72). The polynucleotidesof the invention may be used in immunization or in production ofimmunogenic viral polypeptides and viral-like particles (VLPs).Immunogenic compositions may comprise one or more polynucleotides,polypeptides, peptides, VLPs, and/or adjuvants as described herein.Particularly preferred are immunogenic compositions including all orcomponents of all the pathogenic Noroviruses and/or Saporoviruses. Inaddition, antigens, other than Norovirus or Sapovirus antigens, may beused in immunogenic compositions (e.g., combination vaccines). Forexample, immunogenic compositions may comprise other antigens that canbe used in immunization against pathogens that cause diarrheal diseases,such as antigens derived from rotavirus.

The invention also provides various processes:

In one embodiment, the invention provides a process for producing apolypeptide of the invention, comprising the step of culturing a hostcell transformed with a nucleic acid of the invention under conditionswhich induce polypeptide expression. By way of example, a Norovirus orSapovirus protein may be expressed by recombinant technology and used todevelop an immunogenic composition comprising a recombinant subunitNorwalk or Norwalk related vaccine. Alternatively the viral capsidprotein genes may also be used to prepare Virus-like particles (VLPs) inyeast cells or using baculovirus/insect cell methodology or VEE/SINalphavirus methodology.

The invention provides a process for producing a polypeptide of theinvention, comprising the step of synthesising at least part of thepolypeptide by chemical means.

The invention provides a process for producing nucleic acid of theinvention, wherein the nucleic acid is prepared (at least in part) bychemical synthesis.

The invention provides a process for producing nucleic acid of theinvention, comprising the step of amplifying nucleic acid using aprimer-based amplification method (e.g. PCR).

The invention provides a process for producing a protein complex of theinvention, comprising the step of contacting a class I MHC protein witha polypeptide of the invention, or a fragment thereof.

The invention provides a process for producing a protein complex of theinvention, comprising the step of administering a polypeptide of theinvention, or a fragment thereof, to a subject. The process may comprisethe further step of purifying the complex from the subject.

The invention provides a process for producing a composition comprisingadmixing a polypeptide and/or a nucleic acid of the invention with apharmaceutically acceptable carrier or diluent.

Thus, the subject invention is represented by, but not limited to, thefollowing numbered embodiments:

1. A polynucleotide comprising the nucleotide sequence of SEQ ID NO:1.

2. A polynucleotide comprising the nucleotide sequence of SEQ ID NO:2.

3. A recombinant polynucleotide comprising a promoter operably linked toa polynucleotide of either embodiment 1 or 2.

4. The recombinant polynucleotide of embodiment 3, wherein said promoteris a hybrid ADH2/GAPDH promoter.

5. The recombinant polynucleotide of embodiment 3, further comprising analpha-factor terminator.

6. The recombinant polynucleotide of embodiment 3, further comprising apolynucleotide encoding an adjuvant operably linked to a promoter.

7. A recombinant polynucleotide comprising a sequence encoding aNorovirus or Sapovirus antigen and a sequence encoding an adjuvantoperably linked to a promoter.

8. The recombinant polynucleotide of either embodiment 6 or 7, whereinsaid adjuvant is a detoxified mutant of an E. coli heat-labile toxin(LT) selected from the group consisting of LT-K63 and LT-R72.

9. The recombinant polynucleotide of embodiment 8 comprising apolynucleotide selected from the group consisting of:

-   -   a) a polynucleotide comprising the sequence of SEQ ID NO:1,    -   b) a polynucleotide comprising a sequence at least 90% identical        to the sequence of SEQ ID NO:1 that is capable of producing        viral-like particles,    -   c) a polynucleotide comprising the sequence of SEQ ID NO:2,    -   d) a polynucleotide comprising a sequence at least 90% identical        to the sequence of SEQ ID NO:2 that is capable of producing        viral-like particles, a polynucleotide encoding a polypeptide        comprising the sequence of SEQ ID NO:3,    -   e) a polynucleotide encoding a polypeptide comprising a sequence        at least 90% identical to the sequence of SEQ ID NO:3 that is        capable of eliciting an immune response against Norwalk virus        major capsid protein, a polynucleotide encoding a polypeptide        comprising the sequence of SEQ ID NO:4, and    -   h) a polynucleotide encoding a polypeptide comprising a sequence        at least 90% identical to the sequence of SEQ ID NO:4 that is        capable of eliciting an immune response against Norwalk virus        minor structural protein.

10. The recombinant polynucleotide of embodiment 8 comprising apolynucleotide selected from the group consisting of:

-   -   a) a polynucleotide encoding a polypeptide comprising at least        one sequence selected from the group consisting of SEQ ID        NOS:3-12, SEQ ID NOS:14-17, and SEQ ID NO:19,    -   b) a polynucleotide encoding a polypeptide comprising at least        one sequence at least 90% identical to a sequence selected from        the group consisting of SEQ ID NOS:3-12, SEQ ID NOS:14-17, and        SEQ ID NO:19 that is capable of eliciting an immune response        against a Norovirus or Sapovirus, and    -   c) a fragment of a polynucleotide of a) or b) comprising a        sequence encoding an immunogenic fragment that is capable of        eliciting an immune response against a Norovirus or Sapovirus.

11. A composition, comprising the recombinant polynucleotide of any ofembodiments 3-10 and a pharmaceutically acceptable excipient.

12. The composition of embodiment 11, further comprising an adjuvant.

13. The composition of embodiment 12, wherein said adjuvant is selectedfrom the group consisting of LT-K63, LT-R72, MF59, and alum.

14. The composition of any one of embodiments 11-13, further comprisinga polynucleotide comprising a sequence encoding an adjuvant.

15. The composition of embodiment 14, wherein said adjuvant is LT-K63 orLT-R72.

16. The composition of any of embodiments 11-15, further comprising amicroparticle.

17. The composition of embodiment 16, wherein said microparticle is apoly(L-lactide), poly(D,L-lactide) or poly(D,L-lactide-co-glycolide)microparticle.

18. The composition of any of embodiments 11-17, further comprisingchitosan.

19. The composition of any of embodiments 11-17, further comprising apolypeptide from a Norovirus or Sapovirus.

20. The composition of embodiment 19, comprising a polypeptide selectedfrom the group consisting of:

-   -   a) a polypeptide comprising a sequence selected from the group        consisting of SEQ ID NOS:3-12, SEQ ID NOS:14-17, and SEQ ID        NO:19,    -   b) a polypeptide comprising a sequence at least 90% identical to        a sequence selected from the group consisting of SEQ ID        NOS:3-12, SEQ ID NOS:14-17, and SEQ ID NO:19, and    -   c) an immunogenic fragment of a polypeptide of a) or b).

21. The composition of embodiment 19, comprising at least twopolypeptides from different isolates of Norovirus or Sapovirus.

22. The composition of embodiment 21, wherein at least one polypeptideis from a virus selected from the group consisting of Norwalk virus(NV), Snow Mountain virus (SMV), and Hawaii virus (HV).

23. The composition of embodiment 22, comprising an NV polypeptide, anSMV polypeptide, and an HV polypeptide.

24. The composition of any of embodiments 11-23, further comprising aviral-like particle from a Norovirus or Sapovirus.

25. The composition of any of embodiments 11-24, further comprising apolynucleotide comprising an ORF1 sequence from a Norovirus orSapovirus.

26. The composition of any of embodiments 11-25, further comprising apolynucleotide comprising an ORF2 sequence from a Norovirus orSapovirus.

27. The composition of any of embodiments 11-26, further comprising apolynucleotide comprising an ORF3 sequence from a Norovirus.

28. A cell transformed with the recombinant polynucleotide of any ofembodiments 3-10.

29. A composition comprising at least two polypeptides from two or morestrains of Norovirus or Sapovirus.

30. The composition of claim 29 comprising at least two capsidpolypeptides from two or more strains of Norovirus or Sapovirus.

31. The composition of embodiment 29 or 30, comprising a polypeptideselected from the group consisting of:

-   -   a) a polypeptide comprising a sequence selected from the group        consisting of SEQ ID NOS:3-12,    -   b) a polypeptide comprising a sequence at least 90% identical to        a sequence selected from the group consisting of SEQ ID        NOS:3-12, and    -   c) an immunogenic fragment of a polypeptide of a) or b).

32. The composition of embodiment 30, wherein at least one capsidpolypeptide is from a virus selected from the group consisting ofNorwalk virus (NV), Snow Mountain virus (SMV), and Hawaii virus (HV).

33. The composition of embodiment 32, comprising an NV ORF2-encodedpolypeptide, an SMV ORF2-encoded polypeptide, and an HV ORF2-encodedpolypeptide.

34. The composition of any of embodiments 31-33, further comprising aSapovirus capsid polypeptide.

35. The composition of any of embodiments 29-34, further comprising apolypeptide encoded by ORF1 from a Norovirus or Sapovirus.

36. The composition of any of embodiments 29-35, further comprising amultiepitope fusion protein comprising at least two polypeptides fromone or more Norovirus or Sapovirus isolates.

37. The composition of embodiment 36, wherein the fusion proteincomprises polypeptides from the same Norovirus or Sapovirus isolate.

38. The composition of embodiment 36, wherein the fusion proteincomprises at least two polypeptides from different Norovirus orSapovirus isolates.

39. The composition of embodiment 36, wherein the fusion proteincomprises sequences that are not in the order in which they occurnaturally in the Norovirus or Sapovirus polyprotein.

40. The composition of any of embodiments 29-39, further comprising anORF1-encoded polyprotein of a Norovirus or Sapovirus or a fragmentthereof.

41. The composition of any of embodiments 29-40, further comprising apolypeptide encoded by ORF3 from a Norovirus.

42. The composition of embodiment 41, comprising a polypeptide selectedfrom the group consisting of:

-   -   a) a polypeptide comprising a sequence selected from the group        consisting of SEQ ID NO:4, SEQ ID NO:7, and SEQ ID NO:9;    -   b) a polypeptide comprising a sequence at least 90% identical to        a sequence selected from the group consisting of SEQ ID NO:4,        SEQ ID NO:7, and SEQ ID NO:9 that is capable of eliciting an        immune response against a Norovirus; and    -   c) an immunogenic fragment of a polypeptide of a) or b) that is        capable of eliciting an immune response against a Norovirus.

43. The composition of any of embodiments 29-42, further comprising avirus-like particle (VLP).

44. The composition of any of embodiments 29-42, further comprising oneor more adjuvants.

45. The composition of embodiment 44, wherein the one or more adjuvantsare selected from the group consisting of LT-K63, LT-R72, MF59, andalum.

46. The composition of any of embodiments 29-45, further comprising amicroparticle.

47. The composition of embodiment 46, wherein said microparticle is apoly(L-lactide), poly(D,L-lactide) or poly(D,L-lactide-co-glycolide)microparticle.

48. The composition of any of embodiments 29-47 comprising all orcomponents of all pathogenic Noroviruses.

49. The composition of any of embodiments 29-47 comprising all orcomponents of all pathogenic Sapoviruses.

50. The composition of any of embodiments 29-47 comprising all orcomponents of all pathogenic Noroviruses and Sapoviruses.

51. A composition comprising virus-like particles (VLPs) comprising atleast two antigens from different strains of Norovirus or Sapovirus.

52. The composition of embodiment 51, wherein at least one antigen isfrom a virus selected from the group consisting of Norwalk virus (NV),Snow Mountain virus (SMV), and Hawaii virus (HV).

53. The composition of embodiment 52, comprising an NV antigen, an SMVantigen, and an HV antigen.

54. The composition of any of embodiments 29-53, further comprising apolynucleotide comprising an ORF2 sequence of a Norovirus or Sapovirus.

55. The composition of embodiment 54, wherein the polynucleotidecomprises the sequence of SEQ ID NO:1 or a sequence at least 90%identical to SEQ ID NO:1.

56. The composition of any of embodiments 29-55, further comprising apolynucleotide comprising an ORF1 sequence of a Norovirus or Sapovirus.

57. The composition of any of embodiments 29-56, further comprising apolynucleotide comprising an ORF3 sequence of a Norovirus.

58. The composition of embodiment 57, wherein the polynucleotidecomprises the sequence of SEQ ID NO:2 or a sequence at least 90%identical to SEQ ID NO:2.

59. A method for producing viral-like particles (VLPs), the methodcomprising:

-   -   a) transforming a host cell with an expression vector comprising        the sequence of SEQ ID NO:1 or SEQ ID NO:2;    -   b) culturing the transformed host cell under conditions whereby        capsid proteins are expressed and assembled into VLPs.

60. A method for producing viral-like particles (VLPs) from more thanone Norovirus or Sapovirus isolate, the method comprising:

-   -   a) transforming a host cell with one or more expression vectors        comprising sequences encoding capsid proteins from more than one        Norovirus or Sapovirus isolate;    -   b) culturing the transformed host cell under conditions whereby        said capsid proteins are expressed and assembled into VLPs.

61. The method of either embodiment 59 or 60, further comprisingtransforming said host cell with an expression vector comprising one ormore sequences encoding a structural protein from a Norovirus orSapovirus.

62. The method of embodiment 61, comprising transforming said host cellwith an expression vector comprising an ORF3 sequence from a Norovirus.

63. The method of embodiment 60, wherein said expression vectorcomprises the nucleotide sequence of SEQ ID NO:2.

64. The method of embodiment 60, wherein said expression vectorcomprises a nucleotide sequence at least 90% identical to SEQ ID NO:2that is capable of producing viral-like particles.

65. The method of any of embodiments 59-64, wherein said expressionvector further comprises one or more ORF1 sequences from a Norovirus orSapovirus.

66. The method of any of embodiments 59-65, further comprisingtransforming a host cell with an expression vector comprising a sequenceencoding an adjuvant.

67. The method of embodiment 63, wherein said adjuvant is a detoxifiedmutant of an E. coli heat-labile toxin (LT) selected from the groupconsisting of LT-K63 and LT-R72.

68. A method for producing a mosaic VLP comprising capsid proteins fromat least two viral strains of Norovirus or Sapovirus, the methodcomprising:

-   -   a) cloning polynucleotides encoding said capsid proteins into        expression vectors; and    -   b) expressing said vectors in the same host cell under        conditions whereby said capsid proteins are expressed and        assembled together into said VLP.

69. The method of any of embodiments 59-68, wherein the host cell is ayeast cell.

70. The method of embodiment 69 wherein the yeast is Saccharomycescerevisiae.

71. The method of any of embodiments 59-68, wherein the host cell is aninsect cell.

72. The method of embodiment 71, wherein the expression vector is abaculovirus vector.

73. The method of any of embodiments 59-68, wherein the expressionvector is an alphavirus vector.

74. The composition of any one of embodiments 11-27 and 29-58, furthercomprising an antigen that is not a Norovirus or Sapovirus antigen.

75. The composition of embodiment 74, wherein the antigen is useful in apediatric vaccine.

76. The composition of embodiment 74, wherein the antigen is useful in avaccine designed to protect elderly or immunocompromised individuals.

77. The composition of embodiment 74, wherein the antigen elicits animmune response against a pathogen that causes diarrheal diseases.

78. The composition of embodiment 77, wherein the antigen is a rotavirusantigen.

79. A method of eliciting an immunological response in a subject,comprising administering the composition of any one of embodiments11-27, 29-58, and 74-78 to said subject.

80. The method of embodiment 79, further comprising administering anadjuvant.

81. The method of embodiment 79 comprising administering saidimmunogenic composition to said subject topically.

82. The method of embodiment 79 comprising administering saidimmunogenic composition to said subject parenterally.

83. The method of embodiment 82, further comprising administering anadjuvant selected from the group consisting of MF59 and alum.

84. The method of embodiment 79 comprising administering saidimmunogenic composition to said subject mucosally.

85. The method of embodiment 84, further comprising administering anadjuvant comprising a detoxified mutant of an E. coli heat-labile toxin(LT) selected from the group consisting of LT-K63 and LT-R72.

86. The method of embodiment 79 comprising the following steps:

-   -   a) mucosally administering a first immunogenic composition        comprising one or more Norovirus or Sapovirus antigens; and    -   b) topically or parenterally administering a second immunogenic        composition comprising one or more Norovirus or Sapovirus        antigens.

87. The method of embodiment 86, wherein the one or more antigens isselected from the group consisting of a Norwalk virus (NV) antigen, aSnow Mountain virus (SMV) antigen, and a Hawaii virus (HV) antigen.

88. The method of embodiment 86, wherein the first immunogeniccomposition is the immunogenic composition of any of embodiments 11-27,29-58, and 74-78.

89. The method of embodiment 86, wherein the second immunogeniccomposition is the immunogenic composition of any of embodiments 11-27,29-58, and 74-78.

90. The method of embodiment 86, wherein the first immunogeniccomposition and the second immunogenic composition are the same.

91. The method of embodiment 86, wherein the first immunogeniccomposition and the second immunogenic composition are different.

92. The method of embodiment 86, wherein step (a) is performed two ormore times.

93. The method of embodiment 86, wherein step (b) is performed two ormore times.

94. The method of embodiment 86, wherein the mucosal administration isintranasal.

95. The method of embodiment 86, wherein the mucosal administration isoral.

96. The method of embodiment 86, wherein the mucosal administration isintrarectal.

97. The method of embodiment 86, wherein the mucosal administration isintravaginal.

98. The method of embodiment 86, where in the parenteral administrationis transcutaneous.

99. A method for treating an infection by a Norovirus or Sapovirus, themethod comprising administering to a subject in need thereof atherapeutically effective amount of the immunogenic composition of anyof embodiments 11-27, 29-58, and 74-78.

100. The method of embodiment 99, wherein multiple therapeuticallyeffective doses of the immunogenic composition are administered to saidsubject.

101. The method of embodiment 100, comprising the following steps:

-   -   a) mucosally administering a therapeutically effective amount of        a first immunogenic composition comprising one or more Norovirus        or Sapovirus antigens; and    -   b) topically or parenterally administering a therapeutically        effective amount of a second immunogenic composition comprising        one or more Norovirus or Sapovirus antigens.

102. The method of embodiment 101, wherein one or more antigens isselected from the group consisting of a Norwalk virus (NV) antigen, aSnow Mountain virus (SMV) antigen, and a Hawaii virus (HV) antigen.

103. The method of embodiment 101, wherein the first immunogeniccomposition is the immunogenic composition of any of embodiments 11-27,29-58, and, 74-78.

104. The method of embodiment 101, wherein the second immunogeniccomposition is the immunogenic composition of any of embodiments 11-27,29-58, and 74-78.

105. The method of embodiment 101, wherein the first immunogeniccomposition and the second immunogenic composition are the same.

106. The method of embodiment 101, wherein the first immunogeniccomposition and the second immunogenic composition are different.

107. The method of embodiment 101, wherein step (a) is performed two ormore times.

108. The method of embodiment 101, wherein step (b) is performed two ormore times.

109. The method of embodiment 101, wherein the mucosal administration isintranasal.

110. The method of embodiment 101, wherein the mucosal administration isoral.

111. The method of embodiment 101, wherein the mucosal administration isintrarectal.

112. The method of embodiment 101, wherein the mucosal administration isintravaginal.

113. The method of embodiment 101, where in the parenteraladministration is transcutaneous.

114. A method for treating an infection by a pathogen that causesdiarrheal diseases, the method comprising administering to a subject inneed thereof a therapeutically effective amount of the immunogeniccomposition of embodiment 77.

115. The method of embodiment 114, wherein multiple therapeuticallyeffective doses of the immunogenic composition are administered to saidsubject.

116. The method of embodiment 115, comprising the following steps:

-   -   a) mucosally administering a therapeutically effective amount of        a first immunogenic composition comprising one or more Norovirus        or Sapovirus antigens; and    -   b) topically or parenterally administering a therapeutically        effective amount of a second immunogenic composition comprising        one or more Norovirus or Sapovirus antigens.

117. The method of any of embodiments 114-116, wherein one or moreantigens is selected from the group consisting of a Norwalk virus (NV)antigen, a Snow Mountain virus (SMV) antigen, and a Hawaii virus (HV)antigen.

118. The method of embodiment 117, wherein the immunogenic compositioncomprises a rotavirus antigen.

119. A method of assessing efficacy of a therapeutic treatment of asubject infected by a Norovirus or Sapovirus, the method comprising:

-   -   a) administering to a subject in need thereof a therapeutically        effective amount of the immunogenic composition of any of        embodiments 11-27, 29-58, and 74-78; and    -   b) monitoring the subject for infection by the Norovirus or        Sapovirus after administration of the composition.

120. A method of assessing efficacy of a prophylactic treatment of asubject, the method comprising:

-   -   a) administering to a subject in need thereof a therapeutically        effective amount of the immunogenic composition of any of        embodiments 11-27, 29-58, and 74-78; and    -   b) monitoring the subject for an immune response against one or        more antigens in the composition after administration of the        composition.

These and other embodiments of the subject invention will readily occurto those of skill in the art in view of the disclosure herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C depict an alignment of the nucleotide sequence of NorWalkvirus (SEQ ID NO:20), including orf2 and orf3 regions (GenBank AccessionNo. M87661, Mar. 26, 1997) and the nucleotide sequence of SEQ ID NO:2(NV.orf2+3), comprising modified orf2 and orf3 sequences. The positionsof sequence modifications in SEQ ID NO:2 are highlighted.

FIGS. 2A-2F depict a translation of the nucleotide sequence of SEQ IDNO:2. FIGS. 2A-2D show the translated amino acid sequence encoded byorf2 (SEQ ID NO:3) and FIGS. 2E-2F show the translated amino acidsequence encoded by orf3 (SEQ ID NO:4).

FIG. 3 depicts a schematic diagram illustrating the generation ofoligonucleotide fragments for assembly of the NV.orf2 and NV.orf2+3constructs. The sequence of SEQ ID NO:2 was divided into four domains asdescribed in Example 1. Oligonucleotides for each of the four domainswere engineered to include EcoR1 and SalI sites at their 5′ and 3′ endsand ligated into a pUC19 subcloning vector cut with the restrictionenzymes EcoR1 and SalI. Further digests with the indicated restrictionenzymes produced the oligonucleotide fragments as shown.

FIG. 4 depicts a schematic diagram illustrating the assembly of theNV.orf2 construct from oligonucleotide fragments. The full-lengthNV.orf2 construct was assembled from four oligonucleotide fragmentsproduced from a series of digests with restriction enzymes as shown. Allfour fragments were gel purified and ligated into the pSP72 vector cutwith the restriction enzymes HindIII and SalI, to create a 1613 basepair (bp) HindIII-SalI insert for the coding sequence of NV.orf2.

FIG. 5 depicts a schematic diagram illustrating the assembly of theNV.orf2+3 construct from oligonucleotide fragments. The full-lengthNV.orf2+3 construct was assembled by ligating the HindIII/XbaI,XbaI/PciI, and PciI/AseI fragments shown with a 595 bp gel purifiedfragment obtained from digesting pUC19.NV.3p #22 with AseI and BspE1,and a gel purified BspEI/SalI fragment of 715 bp, obtained frompUC19.NV.orf3 #31, into the pSP72 HindIII/SalI vector (see Example 1).

FIG. 6 depicts a schematic diagram illustrating the subcloning of thefull-length pSP72.NV.orf2 #1 into the pBS24.1 vector to produce thepd.NV.orf2#1 construct for expression in yeast. A 1613 bp NV.orf2fragment, obtained by digestion with the restriction enzymes HindIII andSalI, was gel isolated and purified. This fragment was ligated with theBamHI/HindIII ADH2/GAPDH yeast hybrid promoter of 1366 bp into thepBS24.1 BamHI/SalI yeast expression vector, as described in Example 1.

FIG. 7 depicts a schematic diagram illustrating the subcloning of thefull-length pSP72.NV.orf2+3 #16 into the pBS24.1 vector to produce thepd.NV.orf2+3#12 construct for expression in yeast. A 2314 bp NV.orf2+3fragment, obtained by digestion with the restriction enzymes HindIII andSalI, was gel isolated and purified. This fragment was ligated with theBamHI/HindIII ADH2/GAPDH yeast hybrid promoter of 1366 bp into thepBS24.1 BamHI/SalI yeast expression vector, as described in Example 1.

FIG. 8 depicts results from expression of recombinant Norwalk virusantigens in yeast. The expression plasmids, pd.NV.orf2 #1 andpd.NV.orf2+3 #12, were expressed in S. cerevisiae strain AD3 [matα,leu2Δ, trp1, ura3-52, prb-1122, pep4-3, prc1-407, cir°, trp+,::DM15[GAP/ADR]. Cell lysates were subjected to sucrose gradientsedimentation, and the recombinant proteins in collected fractions weredetected using the RIDASCREEN Norovirus immunoassay (SciMedxCorporation).

FIG. 9 shows an electron micrograph of recombinant Norovirus particlesproduced by expression of pd.NV.orf2+3 #12 in yeast.

FIG. 10 depicts a schematic diagram illustrating the subcloning of thefull-length NV.orf2 and NV.orf2+3 into the PCET906A shuttle vector. A1534 bp KpnI/SalI NV.orf2 fragment and a 2235 bp KpnI/SalI NV.orf2+3fragment were isolated by digesting pSP72.NV.orf2 #1 and pSP72.NV.orf2+3#16, respectively, with KpnI and SalI. The gel purified KpnI/SalINV.orf2 and KpnI/SalI NV.orf2+3 fragments were ligated with a 63 bpsynthetic oligo that included an NheI site at the beginning, a sequenceencoding amino acids 1-21 of the capsid protein, and a KpnI site at theend and cloned into the PCET906A NheI/SalI v. shuttle vector (ML Labs).

FIG. 11 depicts a schematic diagram illustrating the subcloning of thefull-length NV.orf2 and NV.orf2+3 into the PBLUEBAC4.5 baculovirusexpression vector. Clones pCET906A.TPA_(L).orf2 #21 andpCET906A.TPA_(L).orf2+3 #34 were digested with NheI and SalI to gelisolate a 1602 bp fragment coding for NV.orf2 and a 2303 bp fragmentcoding for NV.orf2+3, respectively. Each of the orf2 and orf2+3NheI/SalI fragments was ligated into the PBLUEBAC4.5 NheI/SalI insectcell expression vector (Invitrogen), creating the plasmidsPBLUEBAC4.5.NV.orf2 #2 and PBLUEBAC4.5.NV.orf2+3 #12.

FIG. 12 depicts results from expression of recombinant Norwalk virusantigens in SF9 insect cells infected with baculovirus. Cell lysateswere subjected to sucrose gradient sedimentation, and the recombinantproteins in collected fractions were detected using the RIDASCREENNorovirus immunoassay (SciMedx Corporation).

FIG. 13 shows an electron micrograph of recombinant Norovirus particlesproduced by expression of PBLUEBAC4.5.NV.orf2+3 #12 in SF9 insect cells.

FIGS. 14A and 14B show the nucleotide sequence of SEQ ID NO:1 (NV.orf2).

FIGS. 15A and 15B show the nucleotide sequence of SEQ ID NO:2(NV.orf2+3).

FIGS. 16A-16I show the ORF1 coding sequence (nucleotides 1-5144 of SEQID NO:13) for the Novirus MD145-12 polyprotein (SEQ ID NO:14) and thedomain boundaries of the polyprotein.

FIGS. 17A-17C show the ORF2 coding sequence (nucleotides 5041-6734 ofSEQ ID NO:13) for the Novirus MD145-12 major capsid protein (SEQ IDNO:21).

FIGS. 18A and 18B show the ORF3 coding sequence (nucleotides 6661-7556of SEQ ID NO:13) for the Novirus MD145-12 minor structural protein (SEQID NO:22).

DETAILED DESCRIPTION

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of pharmacology, chemistry,biochemistry, recombinant DNA techniques and immunology, within theskill of the art. Such techniques are explained fully in the literature.See, e.g., Handbook of Experimental Immunology, Vols. I-IV (D. M. Weirand C. C. Blackwell eds., Blackwell Scientific Publications); A. L.Lehninger, Biochemistry (Worth Publishers, Inc., current addition);Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition,1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., AcademicPress, Inc.).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in theirentireties.

I. DEFINITIONS

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referencesunless the content clearly dictates otherwise. Thus, for example,reference to “a polynucleotide” includes a mixture of two or more suchpolynucleotides, and the like.

The term “comprising” means “including” as well as “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

The term “about” in relation to a numerical value x means, for example,x±10%.

As used herein, the terms “Norovirus” and “Norwalk-like virus” refer tomembers of the genus Norovirus of the family Caliciviridae ofpositive-sense, single-stranded RNA, nonenveloped viruses (Green et al.,Human Caliciviruses, in Fields Virology Vol. 1, pp. 841-874 (Knipe andHowley, editors-in-chief, 4th ed., Lippincott Williams & Wilkins 2001)).The term Norovirus includes strains in all genogroups of the virus.Currently, Norovirus strains are divided into four genogroups (GI-GIV),which are subdivided into at least 20 genetic clusters. In particular,the term Norovirus includes, but is not limited to, the species Norwalkvirus (NV), Lordsdale virus (LV), Mexico virus (MV), Hawaii virus (HV),Snow Mountain virus (SMV), Desert Shield virus (DSV), and Southhamptonvirus (SV). A large number of Norovirus isolates have been partially orcompletely sequenced. See, e.g., the Calicivirus Sequence Database, theNorovirus Database and the GenBank NCBI database. The term Norovirusalso includes isolates not characterized at the time of filing.

As used herein, the terms “Sapovirus” and “Sapporo-like virus” refer tomembers of the genus Sapovirus of the family Caliciviridae ofpositive-sense, single-stranded RNA, nonenveloped viruses (Green et al.,supra). The term Sapovirus includes strains in all genogroups of thevirus. Currently, Sapovirus strains are divided into five genogroups(GI-GV) based on their capsid (VP1) sequences. In particular, the termSapovirus includes, but is not limited to, the species Sapporo virus,London/29845 virus, Manchester virus, Houston/86 virus, Houston/90virus, and Parkville virus. A large number of Sapovirus isolates havebeen partially or completely sequenced. See, e.g., the CalicivirusSequence Database and the GenBank NCBI database. The term Sapovirus alsoincludes isolates not characterized at the time of filing.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation and the like. Furthermore, for purposes ofthe present invention, a “polypeptide” refers to a protein whichincludes modifications, such as deletions, additions and substitutions(generally conservative in nature), to the native sequence, so long asthe protein maintains the desired activity. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts which produce the proteins or errorsdue to PCR amplification.

“Substantially purified” generally refers to isolation of a substance(compound, polynucleotide, protein, polypeptide, polypeptidecomposition) such that the substance comprises the majority percent ofthe sample in which it resides. Typically in a sample, a substantiallypurified component comprises 50%, preferably 80%-85%, more preferably90-95% of the sample. Techniques for purifying polynucleotides andpolypeptides of interest are well-known in the art and include, forexample, ion-exchange chromatography, affinity chromatography andsedimentation according to density.

By “isolated” is meant, when referring to a polypeptide, that theindicated molecule is separate and discrete from the whole organism withwhich the molecule is found in nature or is present in the substantialabsence of other biological macro-molecules of the same type. The term“isolated” with respect to a polynucleotide is a nucleic acid moleculedevoid, in whole or part, of sequences normally associated with it innature; or a sequence, as it exists in nature, but having heterologoussequences in association therewith; or a molecule disassociated from thechromosome.

As used herein, the terms “label” and “detectable label” refer to amolecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzymesubstrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes,metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.The term “fluorescer” refers to a substance or a portion thereof whichis capable of exhibiting fluorescence in the detectable range.Particular examples of labels which may be used under the inventioninclude fluorescein, rhodamine, dansyl, umbelliferone, Texas red,luminol, acradimum esters, NADPH and α-β-galactosidase.

“Homology” refers to the percent identity between two polynucleotide ortwo polypeptide moieties. Two nucleic acid, or two polypeptide sequencesare “substantially homologous” to each other when the sequences exhibitat least about 50% sequence identity, preferably at least about 75%sequence identity, more preferably at least about 80%-85% sequenceidentity, more preferably at least about 90% sequence identity, and mostpreferably at least about 95%-98% sequence identity over a definedlength of the molecules. As used herein, substantially homologous alsorefers to sequences showing complete identity to the specified sequence.

In general, “identity” refers to an exact nucleotide-to-nucleotide oramino acid-to-amino acid correspondence of two polynucleotides orpolypeptide sequences, respectively. Percent identity can be determinedby a direct comparison of the sequence information between two moleculesby aligning the sequences, counting the exact number of matches betweenthe two aligned sequences, dividing by the length of the shortersequence, and multiplying the result by 100. Readily available computerprograms can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M. O. Dayhoff ed., 5Suppl. 3:353-358, National biomedical Research Foundation, Washington,D.C., which adapts the local homology algorithm of Smith and WatermanAdvances in Appl. Math. 2:482-489, 1981 for peptide analysis. Programsfor determining nucleotide sequence identity are available in theWisconsin Sequence Analysis Package, Version 8 (available from GeneticsComputer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAPprograms, which also rely on the Smith and Waterman algorithm. Theseprograms are readily utilized with the default parameters recommended bythe manufacturer and described in the Wisconsin Sequence AnalysisPackage referred to above. For example, percent identity of a particularnucleotide sequence to a reference sequence can be determined using thehomology algorithm of Smith and Waterman with a default scoring tableand a gap penalty of six nucleotide positions.

Another method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages the Smith-Waterman algorithm can beemployed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “Match” value reflects “sequenceidentity.” Other suitable programs for calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs are readily available.

Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single-stranded-specificnuclease(s), and size determination of the digested fragments. DNAsequences that are substantially homologous can be identified in aSouthern hybridization experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization,supra.

“Recombinant” as used herein to describe a nucleic acid molecule means apolynucleotide of genomic, cDNA, viral, semisynthetic, or syntheticorigin which, by virtue of its origin or manipulation, is not associatedwith all or a portion of the polynucleotide with which it is associatedin nature. The term “recombinant” as used with respect to a protein orpolypeptide means a polypeptide produced by expression of a recombinantpolynucleotide. In general, the gene of interest is cloned and thenexpressed in transformed organisms, as described further below. The hostorganism expresses the foreign gene to produce the protein underexpression conditions.

The term “transformation” refers to the insertion of an exogenouspolynucleotide into a host cell, irrespective of the method used for theinsertion. For example, direct uptake, transduction or f-mating areincluded. The exogenous polynucleotide may be maintained as anon-integrated vector, for example, a plasmid, or alternatively, may beintegrated into the host genome.

“Recombinant host cells”, “host cells,” “cells”, “cell lines,” “cellcultures”, and other such terms denoting microorganisms or highereukaryotic cell lines cultured as unicellular entities refer to cellswhich can be, or have been, used as recipients for recombinant vector orother transferred DNA, and include the original progeny of the originalcell which has been transfected.

A “coding sequence” or a sequence which “encodes” a selectedpolypeptide, is a nucleic acid molecule which is transcribed (in thecase of DNA) and translated (in the case of mRNA) into a polypeptide invivo when placed under the control of appropriate regulatory sequences(or “control elements”). The boundaries of the coding sequence can bedetermined by a start codon at the 5′ (amino) terminus and a translationstop codon at the 3′ (carboxy) terminus. A coding sequence can include,but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA,genomic DNA sequences from viral or procaryotic DNA, and even syntheticDNA sequences. A transcription termination sequence may be located 3′ tothe coding sequence.

Typical “control elements,” include, but are not limited to,transcription promoters, transcription enhancer elements, transcriptiontermination signals, polyadenylation sequences (located 3′ to thetranslation stop codon), sequences for optimization of initiation oftranslation (located 5′ to the coding sequence), and translationtermination sequences.

The term “nucleic acid” includes DNA and RNA, and also their analogues,such as those containing modified backbones (e.g. phosphorothioates,etc.), and also peptide nucleic acids (PNA), etc. The invention includesnucleic acids comprising sequences complementary to those describedabove (e.g. for antisense or probing purposes).

“Operably linked” refers to an arrangement of elements wherein thecomponents so described are configured so as to perform their usualfunction. Thus, a given promoter operably linked to a coding sequence iscapable of effecting the expression of the coding sequence when theproper enzymes are present. The promoter need not be contiguous with thecoding sequence, so long as it functions to direct the expressionthereof. Thus, for example, intervening untranslated yet transcribedsequences can be present between the promoter sequence and the codingsequence and the promoter sequence can still be considered “operablylinked” to the coding sequence.

“Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 to 5 amino acids,more preferably at least 8 to 10 amino acids, and even more preferablyat least 15 to 20 amino acids from a polypeptide encoded by the nucleicacid sequence.

“Expression cassette” or “expression construct” refers to an assemblywhich is capable of directing the expression of the sequence(s) orgene(s) of interest. An expression cassette generally includes controlelements, as described above, such as a promoter which is operablylinked to (so as to direct transcription of) the sequence(s) or gene(s)of interest, and often includes a polyadenylation sequence as well.Within certain embodiments of the invention, the expression cassettedescribed herein may be contained within a plasmid construct. Inaddition to the components of the expression cassette, the plasmidconstruct may also include, one or more selectable markers, a signalwhich allows the plasmid construct to exist as single-stranded DNA(e.g., a M13 origin of replication), at least one multiple cloning site,and a “mammalian” origin of replication (e.g., a SV40 or adenovirusorigin of replication).

“Purified polynucleotide” refers to a polynucleotide of interest orfragment thereof which is essentially free, e.g., contains less thanabout 50%, preferably less than about 70%, and more preferably less thanabout at least 90%, of the protein with which the polynucleotide isnaturally associated. Techniques for purifying polynucleotides ofinterest are well-known in the art and include, for example, disruptionof the cell containing the polynucleotide with a chaotropic agent andseparation of the polynucleotide(s) and proteins by ion-exchangechromatography, affinity chromatography and sedimentation according todensity.

The term “transfection” is used to refer to the uptake of foreign DNA bya cell. A cell has been “transfected” when exogenous DNA has beenintroduced inside the cell membrane. A number of transfection techniquesare generally known in the art. See, e.g., Graham et al. (1973)Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratorymanual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986)Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousDNA moieties into suitable host cells. The term refers to both stableand transient uptake of the genetic material, and includes uptake ofpeptide- or antibody-linked DNAs.

A “vector” is capable of transferring nucleic acid sequences to targetcells (e.g., viral vectors, non-viral vectors, particulate carriers, andliposomes). Typically, “vector construct,” “expression vector,” and“gene transfer vector,” mean any nucleic acid construct capable ofdirecting the expression of a nucleic acid of interest and which cantransfer nucleic acid sequences to target cells. Thus, the term includescloning and expression vehicles, as well as viral vectors.

“ADH II” refers to the glucose-repressible alcohol dehydrogenase II fromyeast, particularly Saccharomyces, and in particular, S. cerevisiae.“ADH2” refers to the yeast gene encoding ADH II, as well as itsassociated regulatory sequences. See, e.g., Russell et al. (1983) J.Biol. Chem. 258:2674-2682.

“UAS” is an art-recognized term for upstream activation sequences orenhancer regions, which are usually short, repetitive DNA sequenceslocated upstream from a promoter's TATA box. Of particular interest inthe present invention is the ADH2 UAS, which is a 22-bp perfect invertedrepeat located upstream from the ADH2 TATA box. See Shuster et al.(1986) Mol. Cell. Biol. 6:1894-1902.

“ADR1” refers to a positive regulatory gene from yeast required for theexpression of ADH II. See, e.g., Denis et al. (1983) Mol. Cell. Biol.3:360-370. The protein encoded by the ADR1 gene is referred to herein as“ADR I”.

By “fragment” is intended a molecule consisting of only a part of theintact full-length sequence and structure. A fragment of a polypeptidecan include a C-terminal deletion, an N-terminal deletion, and/or aninternal deletion of the native polypeptide. A fragment of a polypeptidewill generally include at least about 5-10 contiguous amino acidresidues of the full-length molecule, preferably at least about 15-25contiguous amino acid residues of the full-length molecule, and mostpreferably at least about 20-50 or more contiguous amino acid residuesof the full-length molecule, or any integer between 5 amino acids andthe number of amino acids in the full-length sequence, provided that thefragment in question retains the ability to elicit the desiredbiological response. A fragment of a nucleic acid can include a5′-deletion, a 3′-deletion, and/or an internal deletion of a nucleicacid. Nucleic acid fragments will generally include at least about5-1000 contiguous nucleotide bases of the full-length molecule and mayinclude at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 orat least 500 contiguous nucleotides of the full-length molecule, or anyinteger between 5 nucleotides and the number of nucleotides in thefull-length sequence. Such fragments may be useful in hybridization,amplification, production of immunogenic fragments, or nucleic acidimmunization.

By “immunogenic fragment” is meant a fragment of an immunogen whichincludes one or more epitopes and thus can modulate an immune responseor can act as an adjuvant for a co-administered antigen. Such fragmentscan be identified using any number of epitope mapping techniques, wellknown in the art. See, e.g., Epitope Mapping Protocols in Methods inMolecular Biology, Vol. 66 (Glenn E. Morris, Ed., 1996) Humana Press,Totowa, N.J. For example, linear epitopes may be determined by e.g.,concurrently synthesizing large numbers of peptides on solid supports,the peptides corresponding to portions of the protein molecule, andreacting the peptides with antibodies while the peptides are stillattached to the supports. Such techniques are known in the art anddescribed in, e.g., U.S. Pat. No. 4,708,871; Geysen et al. (1984) Proc.Natl. Acad. Sci. USA 81:3998-4002; Geysen et al. (1986) Molec. Immunol.23:709-715, all incorporated herein by reference in their entireties.Similarly, conformational epitopes are readily identified by determiningspatial conformation of amino acids such as by, e.g., x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, supra. Antigenic regions of proteins can alsobe identified using standard antigenicity and hydropathy plots, such asthose calculated using, e.g., the Omiga version 1.0 software programavailable from the Oxford Molecular Group. This computer program employsthe Hopp/Woods method, Hopp et al., Proc. Natl. Acad. Sci. USA (1981)78:3824-3828 for determining antigenicity profiles, and theKyte-Doolittle technique, Kyte et al., J. Mol. Biol. (1982) 157:105-132for hydropathy plots.

Immunogenic fragments, for purposes of the present invention, willusually be at least about 2 amino acids in length, more preferably about5 amino acids in length, and most preferably at least about 10 to about15 amino acids in length. There is no critical upper limit to the lengthof the fragment, which could comprise nearly the full-length of theprotein sequence, or even a fusion protein comprising two or moreepitopes.

As used herein, the term “epitope” generally refers to the site on anantigen which is recognised by a T-cell receptor and/or an antibody.Preferably it is a short peptide derived from or as part of a proteinantigen. However the term is also intended to include peptides withglycopeptides and carbohydrate epitopes. Several different epitopes maybe carried by a single antigenic molecule. The term “epitope” alsoincludes modified sequences of amino acids or carbohydrates whichstimulate responses which recognise the whole organism. It isadvantageous if the selected epitope is an epitope of an infectiousagent, which causes the infectious disease.

The epitope can be generated from knowledge of the amino acid andcorresponding DNA sequences of the peptide or polypeptide, as well asfrom the nature of particular amino acids (e.g., size, charge, etc.) andthe codon dictionary, without undue experimentation. See, e.g., IvanRoitt, Essential Immunology, 1988; Kendrew, supra; Janis Kuby,Immunology, 1992 e.g., pp. 79-81. Some guidelines in determining whethera protein will stimulate a response, include: Peptide length—preferablythe peptide is about 8 or 9 amino acids long to fit into the MHC class Icomplex and about 13-25 amino acids long to fit into a class II MHCcomplex. This length is a minimum for the peptide to bind to the MHCcomplex. It is preferred for the peptides to be longer than theselengths because cells may cut peptides. The peptide may contain anappropriate anchor motif which will enable it to bind to the variousclass I or class II molecules with high enough specificity to generatean immune response (See Bocchia, M. et al, Specific Binding of LeukemiaOncogene Fusion Protein Pentides to HLA Class I Molecules, Blood85:2680-2684; Englehard, V H, Structure of peptides associated withclass I and class II MHC molecules Ann. Rev. Immunol. 12:181 (1994)).This can be done, without undue experimentation, by comparing thesequence of the protein of interest with published structures ofpeptides associated with the MHC molecules. Thus, the skilled artisancan ascertain an epitope of interest by comparing the protein sequencewith sequences listed in the protein database.

For a description of various Norovirus capsid epitopes, see, e.g., Hardyet al., U.S. Patent Application Publication No. 2005/0152911;incorporated herein by reference in its entirety. In particular, Hardyet al. have identified epitopes of the Norwalk virus capsid protein atresidues 133-137 and of the Snow Mountain virus capsid protein atresidues 319-327, comprising the following sequences: WTRGSHNL (SEQ IDNO:23), WTRGGHGL (SEQ ID NO:24), WTRGQHQL (SEQ ID NO:25), or WLPAPIDKL(SEQ ID NO:26) Immunogenic polypeptides comprising such capsid epitopesand nucleic acids encoding them may be used in the practice of theinvention.

As used herein, the term “T cell epitope” refers generally to thosefeatures of a peptide structure which are capable of inducing a T cellresponse and a “B cell epitope” refers generally to those features of apeptide structure which are capable of inducing a B cell response.

An “immunological response” to an antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto an antigen present in the composition of interest. For purposes ofthe present invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTL”s). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote thedestruction of intracellular microbes, or the lysis of cells infectedwith such microbes. Another aspect of cellular immunity involves anantigen-specific response by helper T-cells. Helper T-cells act to helpstimulate the function, and focus the activity of, nonspecific effectorcells against cells displaying peptide antigens in association with MHCmolecules on their surface. A “cellular immune response” also refers tothe production of cytokines, chemokines and other such moleculesproduced by activated T-cells and/or other white blood cells, includingthose derived from CD4+ and CD8+ T-cells.

A composition or vaccine that elicits a cellular immune response mayserve to sensitize a vertebrate subject by the presentation of antigenin association with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376. Recent methods of measuringcell-mediated immune response include measurement of intracellularcytokines or cytokine secretion by T-cell populations, or by measurementof epitope specific T-cells (e.g., by the tetramer technique)(reviewedby McMichael, A. J., and O'Callaghan, C. A., J. Exp. Med. 187(9)1367-1371, 1998; Mcheyzer-Williams, M. G., et al, Immunol. Rev.150:5-21, 1996; Lalvani, A., et al, J. Exp. Med. 186:859-865, 1997).

Thus, an immunological response as used herein may be one thatstimulates the production of antibodies (e.g., neutralizing antibodiesthat block bacterial toxins and pathogens such as viruses entering cellsand replicating by binding to toxins and pathogens, typically protectingcells from infection and destruction). The antigen of interest may alsoelicit production of CTLs. Hence, an immunological response may includeone or more of the following effects: the production of antibodies byB-cells; and/or the activation of suppressor T-cells and/ormemory/effector T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest. These responses mayserve to neutralize infectivity, and/or mediate antibody-complement, orantibody dependent cell cytotoxicity (ADCC) to provide protection to animmunized host. Such responses can be determined using standardimmunoassays and neutralization assays, well known in the art. (See,e.g., Montefiori et al. (1988) J. Clin Microbiol. 26:231-235; Dreyer etal. (1999) AIDS Res Hum Retroviruses (1999) 15(17):1563-1571). Theinnate immune system of mammals also recognizes and responds tomolecular features of pathogenic organisms via activation of Toll-likereceptors and similar receptor molecules on immune cells. Uponactivation of the innate immune system, various non-adaptive immuneresponse cells are activated to, e.g., produce various cytokines,lymphokines and chemokines. Cells activated by an innate immune responseinclude immature and mature Dendritic cells of the moncyte andplamsacytoid lineage (MDC, PDC), as well as gamma, delta, alpha and betaT cells and B cells and the like. Thus, the present invention alsocontemplates an immune response wherein the immune response involvesboth an innate and adaptive response.

An “immunogenic composition” is a composition that comprises anantigenic molecule where administration of the composition to a subjectresults in the development in the subject of a humoral and/or a cellularimmune response to the antigenic molecule of interest.

The terms “immunogenic” protein or polypeptide refer to an amino acidsequence which elicits an immunological response as described above. An“immunogenic” protein or polypeptide, as used herein, includes thefull-length sequence of the protein in question, including the precursorand mature forms, analogs thereof, or immunogenic fragments thereof.

By “nucleic acid immunization” is meant the introduction of a nucleicacid molecule encoding one or more selected antigens into a host cell,for the in vivo expression of an antigen, antigens, an epitope, orepitopes. The nucleic acid molecule can be introduced directly into arecipient subject, such as by injection, inhalation, oral, intranasaland mucosal administration, or the like, or can be introduced ex vivo,into cells which have been removed from the host. In the latter case,the transformed cells are reintroduced into the subject where an immuneresponse can be mounted against the antigen encoded by the nucleic acidmolecule.

“Gene transfer” or “gene delivery” refers to methods or systems forreliably inserting DNA or RNA of interest into a host cell. Such methodscan result in transient expression of non-integrated transferred DNA,extrachromosomal replication and expression of transferred replicons(e.g., episomes), or integration of transferred genetic material intothe genomic DNA of host cells. Gene delivery expression vectors include,but are not limited to, vectors derived from bacterial plasmid vectors,viral vectors, non-viral vectors, alphaviruses, pox viruses and vacciniaviruses. When used for immunization, such gene delivery expressionvectors may be referred to as vaccines or vaccine vectors.

The term “derived from” is used herein to identify the original sourceof a molecule but is not meant to limit the method by which the moleculeis made which can be, for example, by chemical synthesis or recombinantmeans.

Generally, a viral polypeptide is “derived from” a particularpolypeptide of a virus (viral polypeptide) if it is (i) encoded by anopen reading frame of a polynucleotide of that virus (viralpolynucleotide), or (ii) displays sequence identity to polypeptides ofthat virus as described above.

A polynucleotide “derived from” a designated sequence refers to apolynucleotide sequence which comprises a contiguous sequence ofapproximately at least about 6 nucleotides, preferably at least about 8nucleotides, more preferably at least about 10-12 nucleotides, and evenmore preferably at least about 15-20 nucleotides corresponding, i.e.,identical or complementary to, a region of the designated nucleotidesequence. The derived polynucleotide will not necessarily be derivedphysically from the nucleotide sequence of interest, but may begenerated in any manner, including, but not limited to, chemicalsynthesis, replication, reverse transcription or transcription, which isbased on the information provided by the sequence of bases in theregion(s) from which the polynucleotide is derived. As such, it mayrepresent either a sense or an antisense orientation of the originalpolynucleotide.

A Norovirus or Sapovirus polynucleotide, oligonucleotide, nucleic acid,protein, polypeptide, or peptide, as defined above, is a moleculederived from a Norovirus or Sapovirus, respectively, including, withoutlimitation, any of the various isolates of Norovirus or Sapovirus. Themolecule need not be physically derived from the particular isolate inquestion, but may be synthetically or recombinantly produced.

In particular, the genomes of Norovirus strains contain three openreading frames: ORF1, which is transcribed into a polyprotein, ORF2,which is transcribed into the major capsid protein VP1, and ORF3, whichis transcribed into the minor structural protein VP2. The Noroviruspolyprotein encoded by ORF1 undergoes cleavage by a 3C-like protease toproduce at least six distinct products, an N-terminal protein (Nterm), a2C-like nucleoside triphosphatase (NTPase), p20 or p22 (depending on thegenogroup), virus protein genome-linked (VPg), a 3C-like cysteineprotease (Pro), and an RNA-dependent RNA polymerase (Pol). See, Belliotet al. (2003) J. Virol. 77:10957-10974, herein incorporated by referencein its entirety. The polyprotein comprises these polypeptides in theorder of NH₂-Nterm-NTPase-p20/p22-VPg-Pro-Pol-COOH. In Norovirus strainMD145-12, the boundaries of the polypeptide domains within thepolyprotein are as follows: Nterm at amino acid residues 1-330, NTPaseat amino acid residues 331-696, P20 at amino acid residues 697-875, VPgat amino acid residues 876-1008, protease at amino acid residues1009-1189, and polymerase at amino acid residues 1190-1699. Although,the foregoing numbering is relative to the polyprotein amino acidsequence of Norovirus strain MD145-12 (SEQ ID NO:14), it is to beunderstood that the corresponding amino acid positions in sequencesobtained from other genotypes and isolates of Norovirus are alsointended to be encompassed by the present invention. Any one of thesepolypeptides encoded by ORF1, or the full-length polyprotein, VP1, orVP2, as well as variants thereof, immunogenic fragments thereof, andnucleic acids encoding such polypeptides, variants or immunogenicfragments can be used in the practice of the invention.

The genomes of Sapovirus strains contain either two or three openreading frames. In strains of Sapovirus having two open reading frames,ORF1 encodes a polyprotein comprising both nonstructural and structuralproteins. The capsid protein VP1 is encoded by ORF1 as a component ofthe Sapovirus polyprotein, and the minor structural protein VP10 isencoded by ORF2. In strains of Sapovirus having three open readingframes, a stop codon precedes the coding region for the capsid protein.A polyprotein not including the capsid protein is encoded by ORF1, thecapsid protein VP1 is encoded by ORF2, and the minor structural proteinVP10 is encoded by ORF3.

Cleavage of the Sapovirus strain Mc10 polyprotein (SEQ ID NO:19, GenBankAccession No. AY237420) by a 3C-like protease produces at least tendistinct products, p11, p28, p35 (NTPase), p32, p14 (VPg), p70(Pro-Pol), p60 (VP1). See, Oka et al. (2005) J. Virol. 79:7283-7290,herein incorporated by reference in its entirety. The polyproteincomprises the polypeptides in the order ofNH₂₋-p28-NTPase-p32-VPg-p70(Pro-Pol)-VP1-COOH. The p70 (Pro-Pol) regionof the polyprotein resides at residues 1056-1720, and the VP1 region ofthe polyprotein resides at residues 1721-2278 (numbered relative toSapovirus strain Mc10 (SEQ ID NO:19, GenBank Accession No. AY237420; seeOka et al. (2005) J. Virol. 79:7283-7290 and Oka et al. (2005) Arch.Virol., August 1 electronic publication). Although, the foregoingnumbering is relative to the polyprotein amino acid sequence ofSapovirus strain Mc10 (SEQ ID NO:19), it is to be understood that thecorresponding amino acid positions in sequences obtained from othergenotypes and isolates of Sapovirus are also intended to be encompassedby the present invention. Any one of the polypeptides encoded by ORF1,or the full-length polyprotein, VP1, or VP10, as well as variantsthereof, immunogenic fragments thereof, and nucleic acids encoding suchpolypeptides, variants or immunogenic fragments can be used in thepractice of the invention.

Nucleic acid and protein sequences for a number of Norovirus isolatesare known. Representative Norovirus sequences are presented in FIGS.1A-1C, 2A-2D, 14A-14B, and 15A-15B, and SEQ ID NOS:1-9 and SEQ IDNOS:13-17. Additional representative sequences, including sequences ofORF1, ORF2, ORF3, and their encoded polypeptides from Norovirus isolatesare listed in the National Center for Biotechnology Information (NCBI)database. See, for example, GenBank entries: Norovirus genogroup 1strain Hu/NoV/West Chester/2001/USA, GenBank Accession No. AY502016;Norovirus genogroup 2 strain Hu/NoV/Braddock Heights/1999/USA, GenBankAccession No. AY502015; Norovirus genogroup 2 strainHu/NoV/Fayette/1999/USA, GenBank Accession No. AY502014; Norovirusgenogroup 2 strain Hu/NoV/Fairfield/1999/USA, GenBank Accession No.AY502013; Norovirus genogroup. 2 strain Hu/NoV/Sandusky/1999/USA,GenBank Accession No. AY502012; Norovirus genogroup 2 strainHu/NoV/Canton/1999/USA, GenBank Accession No. AY502011; Norovirusgenogroup 2 strain Hu/NoV/Tiffin/1999/USA, GenBank Accession No.AY502010; Norovirus genogroup 2 strain Hu/NoV/CS-E1/2002/USA, GenBankAccession No. AY50200; Norovirus genogroup 1 strainHu/NoV/Wisconsin/2001/USA, GenBank Accession No. AY502008; Norovirusgenogroup 1 strain Hu/NoV/CS-841/2001/USA, GenBank Accession No.AY502007; Norovirus genogroup 2 strain Hu/NoV/Hiram/2000/USA, GenBankAccession No. AY502006; Norovirus genogroup 2 strainHu/NoV/Tontogany/1999/USA, GenBank Accession No. AY502005; Norwalkvirus, complete genome, GenBank Accession No. NC_(—)001959; NorovirusHu/GI/Otofuke/1979/JP genomic RNA, complete genome, GenBank AccessionNo. AB187514; Norovirus Hu/Hokkaido/133/2003/JP, GenBank Accession No.AB212306; Norovirus Sydney 2212, GenBank Accession No. AY588132; Norwalkvirus strain SN2000JA, GenBank Accession No. AB 190457; Lordsdale viruscomplete genome, GenBank Accession No. X86557; Norwalk-like virusgenomic RNA, Gifu'96, GenBank Accession No. AB045603; Norwalk virusstrain Vietnam 026, complete genome, GenBank Accession No. AF504671;Norovirus Hu/GII.4/2004/NL, GenBank Accession No. AY883096; NorovirusHu/GII/Hokushin/03/JP, GenBank Accession No. AB195227; NorovirusHu/GII/Kamo/03/JP, GenBank Accession No. AB195228; NorovirusHu/GII/Sinsiro/97/JP, GenBank Accession No. AB195226; NorovirusHu/GII/Ina/02/JP, GenBank Accession No. AB195225; NorovirusHu/NLV/GII/Neustrelitz260/2000/DE, GenBank Accession No. AY772730;Norovirus Hu/NLV/Dresden174/pUS-NorII/1997/GE, GenBank Accession No.AY741811; Norovirus Hu/NLV/Oxford/B2S16/2002/UK, GenBank Accession No.AY587989; Norovirus Hu/NLV/Oxford/B4S7/2002/UK, GenBank Accession No.AY587987; Norovirus Hu/NLV/Witney/B7S2/2003/UK, GenBank Accession No.AY588030; Norovirus Hu/NLV/Banbury/B9S23/2003/UK, GenBank Accession No.AY588029; Norovirus Hu/NLV/ChippingNorton/2003/UK, GenBank Accession No.AY588028; Norovirus Hu/NLV/Didcot/B9S2/2003/UK, GenBank Accession No.AY588027; Norovirus Hu/NLV/Oxford/B8S5/2002/UK, GenBank Accession No.AY588026; Norovirus Hu/NLV/Oxford/B6S4/2003/UK, GenBank Accession No.AY588025; Norovirus Hu/NLV/Oxford/B6S5/2003/UK, GenBank Accession No.AY588024; Norovirus Hu/NLV/Oxford/B5S23/2003/UK, GenBank Accession No.AY588023; Norovirus Hu/NLV/Oxford/B6S2/2003/UK, GenBank Accession No.AY588022; Norovirus Hu/NLV/Oxford/B6S6/2003/UK, GenBank Accession No.AY588021; Norwalk-like virus isolate Bo/Thirsk10/00/UK, GenBankAccession No. AY126468; Norwalk-like virus isolate Bo/Penrith55/00/UK,GenBank Accession No. AY126476; Norwalk-like virus isolateBo/Aberystwyth24/00/UK, GenBank Accession No. AY126475; Norwalk-likevirus isolate Bo/Dumfries/94/UK, GenBank Accession No. AY126474;Norovirus NLV/IF2036/2003/Iraq, GenBank Accession No. AY675555;Norovirus NLV/IF1998/2003/Iraq, GenBank Accession No. AY675554;Norovirus NLV/BUDS/2002/USA, GenBank Accession No. AY660568; NorovirusNLV/Paris Island/2003/USA, GenBank Accession No. AY652979; Snow Mountainvirus, complete genome, GenBank Accession No. AY134748; Norwalk-likevirus NLV/Fort Lauderdale/560/1998/US, GenBank Accession No. AF414-426;Hu/Norovirus/hiroshima/1999/JP(9912-02F), GenBank Accession No.AB044366; Norwalk-like virus strain 11MSU-MW, GenBank Accession No.AY274820; Norwalk-like virus strain B-1 SVD, GenBank Accession No.AY274819; Norovirus genogroup 2 strain Hu/NoV/Farmington Hills/2002/USA,GenBank Accession No. AY502023; Norovirus genogroup 2 strainHu/NoV/CS-G4/2002/USA, GenBank Accession No. AY502022; Norovirusgenogroup 2 strain Hu/NoV/CS-G2/2002/USA, GenBank Accession No.AY502021; Norovirus genogroup 2 strain Hu/NoV/CS-G12002/USA, GenBankAccession No. AY502020; Norovirus genogroup 2 strainHu/NoV/Anchorage/2002/USA, GenBank Accession No. AY502019; Norovirusgenogroup 2 strain Hu/NoV/CS-D1/2002/CAN, GenBank Accession No.AY502018; Norovirus genogroup 2 strain Hu/NoV/Germanton/2002/USA,GenBank Accession No. AY502017; Human calicivirusNLV/GII/Langen1061/2002/DE, complete genome, GenBank Accession No.AY485642; Murine norovirus 1 polyprotein, GenBank Accession No.AY228235; Norwalk virus, GenBank Accession No. AB067536; Humancalicivirus NLV/Mex7076/1999, GenBank Accession No. AF542090; Humancalicivirus NLV/Oberhausen 455/01/DE, GenBank Accession No. AF539440;Human calicivirus NLV/Herzberg 385/01/DE, GenBank Accession No.AF539439; Human calicivirus NLV/Boxer/2001/US, GenBank Accession No.AF538679; Norwalk-like virus genomic RNA, complete genome, GenBankAccession No. AB081723; Norwalk-like virus genomic RNA, complete genome,isolate:Saitama U201, GenBank Accession No. AB039782; Norwalk-like virusgenomic RNA, complete genome, isolate:Saitama U18, GenBank Accession No.AB039781; Norwalk-like virus genomic RNA, complete genome,isolate:Saitama U25, GenBank Accession No. AB039780; Norwalk virusstrain:U25GII, GenBank Accession No. AB067543; Norwalk virusstrain:U201GII, GenBank Accession No. AB067542; Norwalk-like virusesstrain 416/97003156/1996/LA, GenBank Accession No. AF080559;Norwalk-like viruses strain 408/97003012/1996/FL, GenBank Accession No.AF080558; Norwalk-like virus NLV/Burwash Landing/331/1995/US, GenBankAccession No. AF414425; Norwalk-like virus NLV/Miami Beach/326/1995/US,GenBank Accession No. AF414424; Norwalk-like virus NLV/WhiteRiver/290/1994/US, GenBank Accession No. AF414423; Norwalk-like virusNLV/New Orleans/306/1994/US, GenBank Accession No. AF414422;Norwalk-like virus NLV/Port Canaveral/301/1994/US, GenBank Accession No.AF414421; Norwalk-like virus NLV/Honolulu/314/1994/US, GenBank AccessionNo. AF414420; Norwalk-like virus NLV/Richmond/283/1994/US, GenBankAccession No. AF414419; Norwalk-like virus NLV/Westover/302/1994/US,GenBank Accession No. AF414418; Norwalk-like virusNLV/UK3-17/12700/1992/GB, GenBank Accession No. AF414417; Norwalk-likevirus NLV/Miami/81/1986/US, GenBank Accession No. AF414416; SnowMountain strain, GenBank Accession No. U70059; Desert Shield virusDSV395, GenBank Accession No. U04469; Norwalk virus, complete genome,GenBank Accession No. AF093797; Hawaii calicivirus, GenBank AccessionNo. U07611; Southampton virus, GenBank Accession No. L07418; Norwalkvirus (SRSV-KY-89/89/J), GenBank Accession No. L23828; Norwalk virus(SRSV-SMA/76/US), GenBank Accession No. L23831; Camberwell virus,GenBank Accession No. U46500; Human calicivirus strain Melksham, GenBankAccession No. X81879; Human calicivirus strain MX, GenBank Accession No.U22498; Minireovirus TV24, GenBank Accession No. U02030; andNorwalk-like virus NLV/Gwynedd/273/1994/US, GenBank Accession No.AF414409; all of which sequences (as entered by the date of filing ofthis application) are herein incorporated by reference. AdditionalNorovirus sequences are disclosed in the following patent publications:WO 05/030806, WO 00/79280, JP2002020399, US2003129588, U.S. Pat. No.6,572,862, WO 94/05700, and WO 05/032457, all of which are hereinincorporated by reference in their entireties. See also Green et al.(2000) J. Infect. Dis. 181 (Suppl. 2):S322-330; Wang et al. (1994) J.Virol. 68:5982-5990; Chen et al. (2004) J. Virol. 78: 6469-6479;Chakravarty et al. (2005) J. Virol. 79: 554-568; and Fankhauser et al.(1998) J. Infect. Dis. 178:1571-1578; for sequence comparisons and adiscussion of genetic diversity and phylogenetic analysis ofNoroviruses.

Nucleic acid and protein sequences for a number of Sapovirus isolatesare also known. Representative Sapovirus sequences are presented in SEQID NOS:10-12. Additional representative sequences, including sequencesof ORF1 and ORF2, and their encoded polypeptides from Sapovirus isolatesare listed in the National Center for Biotechnology Information (NCBI)database. See, for example, GenBank entries: Sapovirus Mc10, GenBankAccession No. NC_(—)010624; Sapporo virus, GenBank Accession No. U65427;Sapovirus Mc10, GenBank Accession No. AY237420; SapovirusSaKaeo-15/Thailand, GenBank Accession No. AY646855; Sapporo virus,GenBank Accession No. NC_(—)006269; Sapovirus C12, GenBank Accession No.NC_(—)006554; Sapovirus C12, GenBank Accession No. AY603425; SapovirusHu/Dresden/pJG-Sap01/DE, GenBank Accession No. AY694184; Humancalicivirus SLV/cruise ship/2000/USA, GenBank Accession No. AY289804;Human calicivirus SLV/Arg39, GenBank Accession No. AY289803; Porcineenteric calicivirus strain LL14, GenBank Accession No. AY425671; Porcineenteric calicivirus, GenBank Accession No. NC_(—)000940; Humancalicivirus strain Mc37, GenBank Accession No. AY237415; Mink entericcalicivirus strain Canada 151A, GenBank Accession No. AY144337; Humancalicivirus SLV/Hou7-1181, GenBank Accession No. AF435814; Humancalicivirus SLV/Mex14917/2000, GenBank Accession No. AF435813; Humancalicivirus SLV/Mex340/1990, GenBank Accession No. AF435812; Porcineenteric calicivirus, GenBank Accession No. AF182760; Sapporovirus-London/29845, GenBank Accession No. U95645; Sapporovirus-Manchester, GenBank Accession No. X86560; Sapporovirus-Houston/86, GenBank Accession No. U95643; Sapporovirus-Houston/90, GenBank Accession No. U95644; and Human calicivirusstrain HuCV/Potsdam/2000/DEU, GenBank Accession No. AF294739; all ofwhich sequences (as entered by the date of filing of this application)are herein incorporated by reference. See also Schuffenecker et al.(2001) Arch Virol.; 146(11):2115-2132; Zintz et al. (2005) Infect.Genet. Evol. 5:281-290; Farkas et al. (2004) Arch. Virol. 149:1309-1323;for sequence comparisons and a discussion of genetic diversity andphylogenetic analysis of Sapoviruses.

As used herein, the terms “major capsid protein” or “major capsidpolypeptide” or “VP1” in reference to a Norovirus refer to a polypeptidecomprising a sequence homologous or identical to the ORF2-encodedpolypeptide of a Norovirus, and includes sequences displaying at leastabout 80-100% sequence identity thereto, including any percent identitywithin these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity thereto.

As used herein, the terms “minor structural protein” or “minorstructural polypeptide” or “VP2” or “small basic protein” in referenceto a Norovirus refer to a polypeptide comprising a sequence homologousor identical to the ORF3-encoded polypeptide of a Norovirus, and includesequences displaying at least about 80-100% sequence identity thereto,including any percent identity within these ranges, such as 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%sequence identity thereto.

As used herein, the terms “capsid protein” or “capsid polypeptide” or“VP1” in reference to a Sapovirus refer to a polypeptide comprising asequence homologous or identical to the capsid polypeptide of aSapovirus, and include sequences displaying at least about 80-100%sequence identity thereto, including any percent identity within theseranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100% sequence identity thereto. The capsidpolypeptide may be encoded by either ORF1 or ORF2 in different strainsof Sapovirus. In some strains, the Sapovirus has two open readingframes: the capsid protein is encoded by ORF1 as part of a polyproteinand a minor structural protein (VP10) is encoded by ORF2. In otherstrains, the Sapovirus has three open reading frames: a stop codonprecedes the coding region for the capsid protein, which is encoded byORF2, and a minor structural protein (VP10) is encoded by ORF3.

As used herein, the terms “minor structural protein” or “minorstructural polypeptide” or “VP10” in reference to a Sapovirus refer to apolypeptide comprising a sequence homologous or identical to thepolypeptide encoded by the open reading frame following the codingregion for the capsid protein in the Sapovirus genome (either ORF2 orORF3 depending on the strain), and include sequences displaying at leastabout 80-100% sequence identity thereto, including any percent identitywithin these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity thereto.

As used herein, the term “Norovirus polyprotein” refers to a polyproteincomprising a sequence homologous or identical to the ORF1-encodedpolyprotein of a Norovirus, and includes sequences displaying at leastabout 80-100% sequence identity thereto, including any percent identitywithin these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity thereto.

As used herein, the term “Sapovirus polyprotein” refers to a polyproteincomprising a sequence homologous or identical to the ORF1-encodedpolyprotein of a Sapovirus, and includes sequences displaying at leastabout 80-100% sequence identity thereto, including any percent identitywithin these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity thereto.

As used herein, the term “virus-like particle” or “VLP” refers to anonreplicating, viral shell, derived from any of several virusesdiscussed further below. A virus-like particle in accordance with theinvention is non replicative and noninfectious because it lacks all orpart of the viral genome, in particular the replicative and infectiouscomponents of the viral genome. VLPs are generally composed of one ormore viral proteins, such as, but not limited to those proteins referredto as capsid, coat, shell, surface, structural proteins (e.g., VP1,VP2), or particle-forming polypeptides derived from these proteins,including the proteins described herein. VLPs can form spontaneouslyupon recombinant expression of capsid proteins in an appropriateexpression system. Methods for producing particular VLPs are known inthe art and discussed more fully below. The presence of VLPs followingrecombinant expression of viral proteins can be detected usingconventional techniques known in the art, such as by electronmicroscopy, biophysical characterization, and the like. For example,VLPs can be isolated by density gradient centrifugation and/oridentified by characteristic density banding. Alternatively,cryoelectron microscopy can be performed on vitrified aqueous samples ofthe VLP preparation in question, and images recorded under appropriateexposure conditions.

As used herein, the term “mosaic VLP” refers to a VLP comprising capsidproteins from more than one type of virus. VLPs which result from intra-and/or inter-capsomeric association of the proteins are included.

By “particle-forming polypeptide” derived from a particular viralprotein is meant a full-length or near full-length viral protein, aswell as a fragment thereof, or a viral protein with internal deletions,which has the ability to form VLPs under conditions that favor VLPformation. Accordingly, the polypeptide may comprise the full-lengthsequence, fragments, truncated and partial sequences, as well as analogsand precursor forms of the reference molecule. The term thereforeintends deletions, additions and substitutions to the sequence, so longas the polypeptide retains the ability to form a VLP. Thus, the termincludes natural variations of the specified polypeptide sincevariations in coat proteins often occur between viral isolates. The termalso includes deletions, additions and substitutions that do notnaturally occur in the reference protein, so long as the protein retainsthe ability to form a VLP. Preferred substitutions are those which areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cystine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids.

An “antigen” refers to a molecule containing one or more epitopes(either linear, conformational or both) that will stimulate a host'simmune-system to make a humoral and/or cellular antigen-specificresponse. The term is used interchangeably with the term “immunogen.”Normally, a B-cell epitope will include at least about 5 amino acids butcan be as small as 3-4 amino acids. A T-cell'epitope, such as a CTLepitope, will include at least about 7-9 amino acids, and a helperT-cell epitope at least about 12-20 amino acids. Normally, an epitopewill include between about 7 and 15 amino acids, such as, 9, 10, 12 or15 amino acids. The term “antigen” denotes both subunit antigens, (i.e.,antigens which are separate and discrete from a whole organism withwhich the antigen is associated in nature), as well as, killed,attenuated or inactivated bacteria, viruses, fungi, parasites or othermicrobes. Antibodies such as anti-idiotype antibodies, or fragmentsthereof, and synthetic peptide mimotopes, which can mimic an antigen orantigenic determinant, are also captured under the definition of antigenas used herein. Similarly, an oligonucleotide or polynucleotide whichexpresses an antigen or antigenic determinant in vivo, such as in genetherapy and DNA immunization applications, is also included in thedefinition of antigen herein.

The term “antibody” encompasses polyclonal and monoclonal antibodypreparations, as well as preparations including hybrid antibodies,altered antibodies, chimeric antibodies and, humanized antibodies, aswell as: hybrid (chimeric) antibody molecules (see, for example, Winteret al. (1991) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab′)₂and F(ab) fragments; Fv molecules (noncovalent heterodimers, see, forexample, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; andEhrlich et al. (1980) Biochem 19:4091-4096); single-chain Fv molecules(sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA85:5879-5883); dimeric and trimeric antibody fragment constructs;minibodies (see, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumberet al. (1992) J Immunology 149B:120-126); humanized antibody molecules(see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al.(1988) Science 239:1534-1536; and U.K. Patent Publication No. GB2,276,169, published 21 Sep. 1994); and, any functional fragmentsobtained from such molecules, wherein such fragments retainspecific-binding properties of the parent antibody molecule.

The terms “hybridize” and “hybridization” refer to the formation ofcomplexes between nucleotide sequences which are sufficientlycomplementary to form complexes via Watson-Crick base pairing. Where aprimer “hybridizes” with target (template), such complexes (or hybrids)are sufficiently stable to serve the priming function required by, e.g.,the DNA polymerase to initiate DNA synthesis.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from a subject, including but not limited to, forexample, blood, plasma, serum, fecal matter, urine, bone marrow, bile,spinal fluid, lymph fluid, samples of the skin, external secretions ofthe skin, respiratory, intestinal, and genitourinary tracts, tears,saliva, milk, blood cells, organs, biopsies and also samples of in vitrocell culture constituents including but not limited to conditioned mediaresulting from the growth of cells and tissues in culture medium, e.g.,recombinant cells, and cell components. In particular, Norovirus orSapovirus may be obtained from biological samples such as vomit ordiarrhea from individuals infected with the viruses.

By “subject” is meant any member of the subphylum chordata, including,without limitation, humans and other primates, including non-humanprimates such as chimpanzees and other apes and monkey species; farmanimals such as cattle, sheep, pigs, goats and horses; domestic mammalssuch as dogs and cats; laboratory animals including rodents such asmice, rats and guinea pigs; birds, including domestic, wild and gamebirds such as chickens, turkeys and other gallinaceous birds, ducks,geese, and the like. The term does not denote a particular age. Thus,both adult and newborn individuals are intended to be covered.

The terms “variant,” “analog” and “mutein” refer to biologically activederivatives of the reference molecule that retain desired activity, suchas antigenic activity in inducing an immune response against Norovirusor Sapovirus. In general, the terms “variant” and “analog” refer tocompounds having a native polypeptide sequence and structure with one ormore amino acid additions, substitutions (generally conservative innature) and/or deletions, relative to the native molecule, so long asthe modifications do not destroy biological activity and which are“substantially homologous” to the reference molecule as defined below.In general, the amino acid sequences of such analogs will have a highdegree of sequence homology to the reference sequence, e.g., amino acidsequence homology of more than 50%, generally more than 60%-70%, evenmore particularly 80%-85% or more, such as at least 90%-95% or more,when the two sequences are aligned. Often, the analogs will include thesame number of amino acids but will include substitutions, as explainedherein: The term “mutein” further includes polypeptides having one ormore amino acid-like molecules including but not limited to compoundscomprising only amino and/or imino molecules, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), polypeptides with substituted linkages, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring (e.g., synthetic), cyclized, branched moleculesand the like. The term also includes molecules comprising one or moreN-substituted glycine residues (a “peptoid”) and other synthetic aminoacids or peptides. (See, e.g., U.S. Pat. Nos. 5,831,005; 5,877,278; and5,977,301; Nguyen et al., Chem. Biol. (2000) 7:463-473; and Simon etal., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for descriptions ofpeptoids). Preferably, the analog or mutein has at least the sameantigenic activity as the native molecule. Methods for makingpolypeptide analogs and muteins are known in the art and are describedfurther below.

As explained above, analogs generally include substitutions that areconservative in nature, i.e., those substitutions that take place withina family of amino acids that are related in their side chains.Specifically, amino acids are generally divided into four families: (1)acidic—aspartate and glutamate; (2) basic—lysine, arginine, histidine;(3) non-polar—alanine, valine, leucine, isoleucine, proline,phenylalanine, methionine, tryptophan; and (4) uncharged polar—glycine,asparagine, glutamine, cysteine, serine threonine, tyrosine.Phenylalanine, tryptophan, and tyrosine are sometimes classified asaromatic amino acids. For example, it is reasonably predictable that anisolated replacement of leucine with isoleucine or valine, an aspartatewith a glutamate, a threonine with a serine, or a similar conservativereplacement of an amino acid with a structurally related amino acid,will not have a major effect on the biological activity. For example,the polypeptide of interest may include up to about 5-10 conservative ornon-conservative amino acid substitutions, or even up to about 15-25conservative or non-conservative amino acid substitutions, or anyinteger between 5-25, so long as the desired function of the moleculeremains intact. One of skill in the art may readily determine regions ofthe molecule of interest that can tolerate change by reference toHopp/Woods and Kyte-Doolittle plots, well known in the art.

The term “multiple epitope fusion antigen” or “multiple epitope fusionprotein” as used herein intends a polypeptide in which multipleNorovirus and/or Sapovirus antigens are part of a single, continuouschain of amino acids, which chain does not occur in nature. TheNorovirus and Sapovirus antigens may be connected directly to each otherby peptide bonds or may be separated by intervening amino acidsequences. The fusion antigens may contain ORF1-encoded, ORF2-encoded,and/or ORF3-encoded polypeptides or fragments thereof, including, forexample, sequences of Norovirus polypeptides, such as N-terminalprotein, NTPase, p20, VPg, protease, polymerase, VP1, and VP2; and/orsequences of Sapovirus polypeptides, such as N-terminal protein, p11,p28, NTPase, p32, VPg, protease, polymerase, VP1, and VP10. The fusionantigens may also contain sequences exogenous to the Norovirus orSapovirus. Moreover, the sequences present may be from multiplegenotypes and/or isolates of Norovirus and Sapovirus.

As used herein, “detoxified” refers to both completely nontoxic and lowresidual toxic mutants of the toxin in question. Toxic protein antigensmay be detoxified where necessary, e.g., detoxification of pertussistoxin by chemical and/or genetic means is known in the art. Preferably,the detoxified protein retains a toxicity of less than 0.01% of thenaturally occurring toxin counterpart, more preferably less than 0.001%and even more preferable, less than 0.0001% of the toxicity of thenaturally occurring toxin counterpart. The toxicity may be measured inmouse CHO cells or preferably by evaluation of the morphological changesinduced in Y1 cells. In particular, Y1 cells are adrenal tumorepithelial cells which become markedly more rounded when treated with asolution containing CT or LT (Ysamure et al., Cancer Res. (1966)26:529-535). The toxicity of CT and LT is correlated with thismorphological transition. Thus, the mutant toxins may be incubated withY1 cells and the morphological changes of the cells assessed.

By “therapeutically effective amount” in the context of the immunogeniccompositions is meant an amount of an immunogen (e.g., immunogenicpolypeptide, fusion protein, polyprotein, VLP, or nucleic acid encodingan antigen) which will induce an immunological response, either forantibody production or for treatment or prevention of Norovirus orSapovirus infection. Such a response will generally result in thedevelopment in the subject of an antibody-mediated and/or a secretory orcellular immune response to the composition. Usually, such a responseincludes but is not limited to one or more of the following effects; theproduction of antibodies from any of the immunological classes, such asimmunoglobulins A, D, E, G or M; the proliferation of B and Tlymphocytes; the provision of activation, growth and differentiationsignals to immunological cells; expansion of helper T cell, suppressor Tcell, and/or cytotoxic T cell and/or γδT cell populations.

For purposes of the present invention; an “effective amount” of anadjuvant will be that amount which enhances an immunological response toa coadministered antigen or nucleic acid encoding an antigen.

As used herein, “treatment” refers to any of (i) the prevention ofinfection or reinfection, as in a traditional vaccine, (ii) thereduction or elimination of symptoms, and (iii) the substantial orcomplete elimination of the pathogen in question. Treatment may beeffected prophylactically (prior to infection) or therapeutically(following infection).

II. MODES OF CARRYING OUT THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified molecules or process parameters as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments of the inventiononly, and is not intended to be limiting. In addition, the practice ofthe present invention will employ, unless otherwise indicated,conventional methods of virology, microbiology, molecular biology,recombinant DNA techniques and immunology all of which are within theordinary skill of the art. Such techniques are explained fully in theliterature. See, e.g., Sambrook, et al., Molecular Cloning: A LaboratoryManual (2nd Edition, 1989); DNA Cloning: A Practical Approach, vol. I &II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); APractical Guide to Molecular Cloning (1984); and Fundamental Virology,2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.). Althougha number of methods and materials similar or equivalent to thosedescribed herein can be used in the practice of the present invention,the preferred materials and methods are described herein.

The present invention includes compositions and methods for immunizing asubject against Norovirus or Sapovirus infection. The invention providesimmunogenic compositions comprising nucleic acids encoding capsidproteins and/or other immunogenic polypeptides from one or more strainsof Norovirus and/or Sapovirus, compositions comprising immunogenicpolypeptides derived from one or more strains of Norovirus and/orSapovirus, compositions comprising VLPs derived from one or more strainsof Norovirus and/or Sapovirus, and compositions comprising mixtures ofsuch immunogenic nucleic acids, polypeptides, and/or VLPs. Nucleic acidsencoding capsid proteins may further be used in the production of VLPs.Such VLPs are useful as vehicles for the presentation of antigens andstimulation of an immune response in a subject to whom the VLPs ornucleic acids encoding such VLPs are administered. Immunogenicpolypeptides to be used in the practice of the invention may includeNorovirus- or Sapovirus-derived polypeptides, including ORF1-encodedpolypeptides, ORF2-encoded polypeptides, ORF3-encoded polypeptides,multiple epitope fusion antigens, and/or ORF1-encoded polyproteins. Inaddition, immunogenic compositions may comprise one or more adjuvants ornucleic acids encoding adjuvants, wherein immunogenic polypeptidesand/or VLPs are mixed or co-expressed with adjuvants. Immunogeniccompositions may also comprise additional antigens other than Norovirusor Sapovirus antigens, such as antigens that can be used in immunizationagainst pathogens that cause diarrheal diseases.

In order to further an understanding of the invention, a more detaileddiscussion is provided below regarding the production of nucleic acids,polypeptides, and VLPs for use in immunogenic compositions and methodsof using such compositions in the treatment or prevention of Norovirusor Sapovirus infection.

A. Polypeptides

Structural Polypeptides, Nonstructural Polypeptides, and Polyproteins

The immunogenic compositions described herein may comprise one or morepolypeptides derived from one or more genotypes and/or isolates ofNorovirus and Sapovirus. Polypeptides that can be used in the practiceof the invention include structural proteins, nonstructural proteins,and polyproteins. Such polypeptides can be full-length proteins orvariants or immunogenic fragments thereof capable of eliciting an immuneresponse to a Norovirus or Sapovirus.

The genomes of Norovirus strains contain three open reading frames:ORF1, comprising approximately 5,000 to 5500 nucleotides, is transcribedinto a 200 kDa polyprotein. ORF2, comprising approximately 1550 to 1650nucleotides, is transcribed into the 60 kDa major capsid protein VP1.ORF3, comprising approximately 1550 to 1650 nucleotides, is transcribedinto the minor structural protein VP2.

The Norovirus polyprotein undergoes cleavage by a 3C-like protease toproduce at least six distinct products, an N-terminal protein (Nterm), a2C-like nucleoside triphosphatase (NTPase), p20 or p22 (depending on thegenogroup), virus protein genome-linked (VPg), a 3C-like cysteineprotease (Pro), and an RNA-dependent RNA polymerase (Pol). See, Belliotet al. (2003) J. Virol. 77:10957-10974, herein incorporated by referencein its entirety. The polyprotein is initially cleaved into the threefragments, Nterm, NTPase, and a p20VPgProPol complex, by the 3C-likeprotease. Further proteolytic processing produces ProPol, P20VPgPro,Pol, P20VPg, VPgPro, p20 and Pro fragments. Completion of polyproteinmaturation, catalyzed by the 3C-like cysteine protease, produces all theseparate polypeptides. The 200 kDa polyprotein comprises thesepolypeptides in the order of NH₂-Nterm-NTPase-p20/p22-VPg-Pro-Pol-COOH.The approximate domain boundaries within the Norovirus polyprotein andthe corresponding nucleotide positions of the ORF1 coding sequence arepresented in Table 1.

TABLE 1 Norovirus Polyprotein Polyprotein Domain ORF1 Coding BoundariesSequence Domain Amino Acid Positions* Nucleotide Positions* Nterm  1-330 5-994 NTPase 331-696  995-2092 P20 697-875 2093-2629 VPg  876-10082630-3028 protease 1009-1189 3029-3271 polymerase 1190-1699 3272-5101*Numbered relative to Norovirus strain MD145-12 (SEQ ID NO: 13, SEQ IDNO: 14, GenBank Accession No. AAK50354). See, Belliot et al. (2003) J.Virol. 77: 10957-10974.

The genomes of Sapovirus strains contain either two or three openreading frames. In strains of Sapovirus having two open reading frames,ORF1 encodes a polyprotein comprising both nonstructural and structuralproteins. The capsid protein VP1 is encoded by ORF1 as a component ofthe Sapovirus polyprotein, and the minor structural protein VP10 isencoded by ORF2. In strains of Sapovirus having three open readingframes, a stop codon precedes the coding region for the capsid protein.A polyprotein not including the capsid protein is encoded by ORF1, thecapsid protein VP1 is encoded by ORF2, and the minor structural proteinVP10 is encoded by ORF3.

Cleavage of the Sapovirus strain Mc10 polyprotein (SEQ ID NO:19, GenBankAccession No. AY237420) by a 3C-like protease produces at least tendistinct products, p11, p28, p35 (NTPase), p32, p14 (VPg), p70(Pro-Pol), p60 (VP1). See, Oka et al. (2005) J. Virol. 79:7283-7290,herein incorporated by reference in its entirety. Initial proteolyticprocessing produces p66 (p28-p35), p46 (p32-p14), and p120 (p32-p14-p70)fragments. The polyprotein comprises the polypeptides in the order ofNH₂-p11-p28-NTPase-p32-VPg-p70(Pro-Pol)-VP1-COOH. The p70 (Pro-Pol)region of the polyprotein resides at residues 1056-1720, and the VP1region of the polyprotein resides at residues 1721-2278 (numberedrelative to Sapovirus strain Mc10 (SEQ ID NO:19, GenBank Accession No.AY237420; see Oka et al. (2005) J. Virol. 79:7283-7290 and Oka et al.(2005) Arch. Virol., August 1 electronic publication).

Nucleic acid and amino acid sequences of a number of Norovirus strainsand isolates, including nucleic acid and amino acid sequences of VP1 andVP2 structural proteins and the various regions of Noroviruspolyproteins, including Nterm, NTPase, p20/p22, VPg, Pro, and Pol genesand polypeptides have been determined. For example, Norwalk virus isdescribed in Jiang et al. (1993) Virology 195:51-61 and Hardy and Estes(1996) Virus Genes 12:287-290; herein incorporated by reference in theirentireties. Snow Mountain virus is described in Lochridge and Hardy(2003) Virus Genes 26:71-82; King and Green (1997) Virus Genes 15:5-7;Wang et al. (1994) J. Virol. 68, 5982-5990; herein incorporated byreference in their entireties. Hawaii virus is described in Lew et al.(1994) J. Infect. Dis. 170:535-542; herein incorporated by reference inits entirety.

Nucleic acid and amino acid sequences of a number of Sapovirus strainsand isolates, including nucleic acid and amino acid sequences of VP1 andVP10 structural proteins and the various regions of Sapoviruspolyproteins, including p11, p28, NTPase, p32, VPg, p70(Pro-Pol), VP1genes and polypeptides have also been determined. For example, Sapporovirus is described in Numata et al. (1997) Arch. Virol. 142:1537-1552;herein incorporated by reference in its entirety. London/29845 virus,Houston/86 virus, and Houston/90 virus are described in Jiang et al.(1997) Arch. Virol. 142:1813-1827; herein incorporated by reference inits entirety. Parkville virus is described in Noel et al. (1997) J. Med.Virol. 52:173-178; herein incorporated by reference in its entirety.

The polypeptides in immunogenic compositions may be encoded by anyregion of a Norovirus or Sapovirus genome. Multiple polypeptides may beincluded in immunogenic compositions. Such compositions may comprisepolypeptides from the same Norovirus or Sapovirus isolate or fromdifferent strains and isolates, including isolates having any of thevarious Norovirus or Sapovirus genotypes, to provide increasedprotection against a broad range of Norovirus and Sapovirus genotypes.Immunogenic compositions may contain both polypeptides derived fromNorovirus strains as well as polypeptides derived from Sapovirusstrains. Multiple viral strains of Norovirus and Sapovirus are known,and multiple polypeptides comprising epitopes derived from any of thesestrains can be used in immunogenic compositions.

The antigens used in the immunogenic compositions of the presentinvention may be present in the composition as individual separatepolypeptides. Generally, the recombinant proteins of the presentinvention are prepared as a GST-fusion protein and/or a His-taggedfusion protein.

Multiepitope Fusion Proteins

The immunogenic compositions described herein may also comprise multipleepitope fusion proteins. See, e.g., International Publication No. WO97/44469, U.S. Pat. No. 6,632,601, U.S. Pat. No. 6,630,298, U.S. Pat.No. 6,514,731, and U.S. Pat. No. 6,797,809; herein incorporated byreference in their entireties. Such fusion proteins include multipleepitopes derived from two or more viral polypeptides of one or moregenotypes and/or isolates of Norovirus and Sapovirus. Multiple epitopefusion proteins offer two principal advantages: first, a polypeptidethat may be unstable or poorly expressed on its own can be assisted byadding a suitable hybrid partner that overcomes the problem; second,commercial manufacture is simplified as only one expression andpurification need be employed in order to produce two polypeptides whichare both antigenically useful.

Multiepitope fusion proteins may contain one or more of the variousdomains of Norovirus or Sapovirus polyproteins (shown in Tables 1 and 2above), full-length polyproteins, VP1 (also referred to herein as acapsid protein), VP2 (also referred to herein as a Norovirus minorstructural protein), and/or VP10 (also referred to herein as a Sapovirusminor structural protein); or fragments thereof, derived from one ormore Norovirus and/or Sapovirus isolates. The polypeptides in fusionproteins may be derived from the same Norovirus or Sapovirus isolate orfrom different strains and isolates, including isolates having any ofthe various Norovirus or Sapovirus genotypes, to provide increasedprotection against a broad range of Norovirus and Sapovirus genotypes.Multiple viral strains of Norovirus and Sapovirus are known, andepitopes derived from any of these strains can be used in a fusionprotein.

It is well known that any given species of organism varies from oneindividual organism to another and further that a given organism such asa virus can have a number of different strains. For example, asexplained above, Norovirus includes at least four genogroups (GI-GIV)and Sapovirus includes at least five genogroups (GI-GV). Each strainincludes a number of antigenic determinants that are in homologousregions present in all strains of Noroviruses or Sapoviruses but areslightly different from one viral strain to another. Thus, a multipleepitope fusion antigen may include multiple polypeptides from differentviral strains of Norovirus or Sapovirus, each comprising a particularhomologous region but each having a different form of an antigenicdeterminant. In general, antigenic determinants may have a high degreeof homology in terms of amino acid sequence, which degree of homology isgenerally 30% or more, preferably 40% or more, when aligned. A fusionprotein may also comprise multiple copies of an epitope, wherein one ormore polypeptides of the fusion protein comprise sequences comprisingexact copies of the same epitope. Additionally, polypeptides can beselected based on the particular viral clades endemic in specificgeographic regions where vaccine compositions containing the fusionswill be used. It is readily apparent that the subject fusions provide aneffective means of treating Norovirus and Sapovirus infection in a widevariety of contexts.

Multiple epitope fusion antigens can be represented by the formulaNH₂-A-{-X-L-}_(n)-B—COOH, wherein: X is an amino acid sequence of aNorovirus or Sapovirus antigen or a fragment thereof; L is an optionallinker amino acid sequence; A is an optional N-terminal amino acidsequence; B is an optional C-terminal amino acid sequence; and n is 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

If an —X— moiety has a leader peptide sequence in its wild-type form,this may be included or omitted in the multiple epitope fusion antigen.In some embodiments, the leader peptides will be deleted except for thatof the —X— moiety located at the N-terminus of the hybrid protein i.e.the leader peptide of X₁ will be retained, but the leader peptides of X₂. . . X_(n) will be omitted. This is equivalent to deleting all leaderpeptides and using the leader peptide of X₁ as moiety -A-.

For each n instances of (—X-L-), linker amino acid sequence -L- may bepresent or absent. For instance, when n=2 the hybrid may beNH₂—X₁-L₁-X₂-L₂-COOH, NH₂—X₁—X₂—COOH, NH₂—X₁-L₁-X₂—COOH,NH₂—X₁—X₂-L₂-COOH, etc. Linker amino acid sequence(s)-L- will typicallybe short, e.g., 20 or fewer amino acids (i.e., 20, 19, 18, 17, 16; 15,14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples include shortpeptide sequences which facilitate cloning, poly-glycine linkers (Gly,where n=2, 3, 4, 5, 6, 7, 8, 9, 10 or more), and histidine tags (His,where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitable linker aminoacid sequences will be apparent to those skilled in the art. A usefullinker is GSGGGG, with the Gly-Ser dipeptide being formed from a BamHIrestriction site, which aids cloning and manipulation, and the (Gly)₄tetrapeptide being a typical poly-glycine linker.

-A- is an optional N-terminal amino acid sequence. This will typicallybe short, e.g., 40 or fewer amino acids (i.e., 40, 39, 38, 37, 36, 35,34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includeleader sequences to direct protein trafficking or short peptidesequences which facilitate cloning or purification (e.g., a histidinetag His_(n) where n=3, 4, 5, 6, 7, 8, 9, 10 or more). Other suitableN-terminal amino acid sequences will be apparent to those skilled in theart. If X₁ lacks its own N-terminus methionine, -A- is preferably anoligopeptide (e.g., with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) whichprovides a N-terminus methionine.

—B— is an optional C-terminal amino acid sequence. This will typicallybe short, e.g., 40 or fewer amino acids (i.e., 40, 39, 38, 37, 36, 35,34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1). Examples includesequences to direct protein trafficking, short peptide sequences whichfacilitate cloning or purification (e.g., His_(n) where n=3, 4, 5, 6, 7,8, 9, 10 or more), or sequences which enhance protein stability. Othersuitable C-terminal amino acid sequences will be apparent to thoseskilled in the art.

The individual antigens of the immunogenic composition within themultiple epitope fusion antigen (individual —X— moieties) may be fromone or more strains or from one or more M types. Where n=2, forinstance, X₂ may be from the same strain or type as X₁ or from adifferent strain or type. Where n=3, the strains might be (i) X₁=X₂=X₃,(ii) X₁≠X₂≠X₃, (iii) X₁≠X₂≠X₃, (iv) X₁≠X₂≠X₃, or (v) X₁=X₃≠X₂, etc.

Where multiple epitope fusion antigens are used, the individual antigenswithin the fusion protein (i.e. individual —X— moieties) may be from oneor more strains. Where n=2, for instance, X₂ may be from the same strainas X₁ or from a different strain. Where n=3, the strains might be (i)X₁=X₂=X₃ (ii) X₁=X₂≠X₃ (iii) X₁≠X₂=X₃ (iv) X₁≠X₂≠X₃ or (v) X₁=X₃≠X₂,etc.

Accordingly, in certain embodiments of the invention antigenicdeterminants from different Norovirus and/or Sapovirus strains may bepresent. Representative multiepitope fusion proteins for use in thepresent invention, comprising polypeptides derived from Norovirus andSapovirus isolates, are discussed below. However, it is to be understoodthat multiepitope fusion proteins comprising other epitopes derived fromNorovirus and Sapovirus genomes or multiepitope fusion proteinscomprising different arrangements of epitopes will also find use inimmunogenic compositions of the invention.

In certain embodiments, the fusion protein comprises one or more capsidand/or minor structural polypeptides from one or more isolates ofNorovirus and/or Sapovirus. In one embodiment, the fusion proteincomprises VP1 polypeptides from more than one Norovirus strain (e.g.,VP1_(NV)-VP1_(SMV), VP1_(NV)-VP1_(SMV)-VP1_(HV),VP1_(NV)-VP1_(SMV)-VP1_(HV)-VP1_(LV), VP1_(SMV)-VP1_(LV)-VP1_(MV),VP1_(NV)-VP1_(SMV)-VP1_(HV)-VP1_(LV)-VP1_(MV)-VP1_(DSV)-VP1_(SV)).

In another embodiment, the fusion protein comprises VP1 polypeptidesfrom more than one Sapovirus strain (e.g.,VP1_(Sapporo)-VP1_(London/29845),VP1_(London/29845)-VP1_(Manchester)-VP1_(Sapporo),VP1_(Manchester)-VP1_(Parkville)-VP1_(Sapporo)-VP1_(London/29845),VP1_(Parkville)-VP1_(Houston/90)-VP1_(Houston/86)-VP1_(Manchester)-VP1_(Sapporo)).

In another embodiment, the fusion protein comprises VP1 polypeptidesfrom Norovirus and Sapovirus strains (e.g.,VP1_(NV)-VP1_(SMV)-VP1_(Sapporo)-VP1_(London/29845),VP1_(Parkville)-VP1_(Houston/90)-VP1_(NV)-VP1_(SMV)-VP1_(HV),VP1_(Manchester) VP1_(NV)-VP1_(SMV)-VP1_(Sapporo)-VP1_(HV), VP1_(LV),VP1_(SMV)-VP1_(Houston/86)VP1_(LV)-VP1_(MV),VP1_(NV)-VP1_(SMV)-VP1_(HV)-VP1_(Sapporo)-VP1_(Houston/90)-VP1_(Houston/86),VP1_(London/29845)-VP1_(LV)-VP1_(MV)-VP1_(DSV)-VP1_(SV)).

In another embodiment, the fusion protein comprises VP2 polypeptidesfrom more than one Norovirus strain (e.g., VP2_(NV)-VP2_(SMV),VP2_(NV)-VP2_(SMV)-VP2_(HV), VP2_(NV)-VP2_(SMV)-VP2_(HV)-VP2_(LV),VP2_(SMV)-VP2_(LV)-VP2_(MV),VP2_(NV)-VP2_(SMV)-VP2_(HV)-VP2_(LV)-VP2_(MV)-VP2_(DSV)-VP2_(SV)).

In another embodiment, the fusion protein comprises VP10 polypeptidesfrom more than one Sapovirus strain (e.g.,VP10_(Sapporo)-VP10_(London/29845),VP10_(London/29845)-VP10_(Manchester)-VP10_(Sapporo),VP10_(Manchester)-VP10_(Parkville)-VP10_(Sapporo)-VP10_(London/29845),VP10_(Parkville)-VP10_(Houston/90)-VP10_(Houston/86)-VP10_(Manchester)-VP10_(Sapporo)).

In another embodiment, the fusion protein comprises VP2 from one ormore. Norovirus strains and VP10 polypeptides from one or more Sapovirusstrains (e.g., VP2_(NV)-VP2_(SMV)-VP10_(Sapporo)-VP10_(London/29845),VP10_(Parkville)-VP10_(Houston/90)-VP2_(NV)-VP2_(SMV)-VP10_(HV),VP10_(Manchester)-VP2_(NV)-VP2_(SMV)-VP10_(Sapporo)-VP2_(HV),VP2_(LV)-VP2_(SMV)-VP10_(Houston/86)-VP2_(LV)-VP2_(MV),VP2_(NV)-VP2_(SMV)-VP2_(HV)-VP10_(Sapporo)-VP10_(Houston/90)-VP10_(Houston/86),VP10_(London/29845)-VP2_(LV)-VP2_(MV)-VP2_(DSV)-VP2_(SV)).

In another embodiment, the fusion protein comprises VP1 and VP2polypeptides from one or more Norovirus strains and VP1 and VP10polypeptides from one or more Sapovirus strains (e.g.,VP1VP2_(NV)-VP1VP10_(London/29845),VP1VP2_(SMV)-VP1VP10_(Houston/86)-VP1VP10_(Houston/90)-VP1VP2_(HV),VP1VP2_(NV)-VP10_(Sapporo)-VP10_(Houston/90)-VP10_(Houston/86)-VP1VP2_(SMV),VP1_(NV)-VP1VP2_(SMV)-VP2_(HV)-VP1_(London/29845),VP1VP2_(NV)-VP10_(Houston/90)-VP1VP10_(Houston/86)-VP1VP2_(SMV)-VP1VP2_(HV)-VP1VP2_(LV),VP1VP2_(SMV)-VP1VP2_(LV)-VP1VP2_(MV)-VP10_(Sapporo)-VP10_(Houston/90)-VP10_(Houston/86),VP1VP2_(LV)-VP1VP2_(MV)-VP10_(Sapporo)-VP10_(London/29845),VP10_(Sapporo)-VP10_(London/29845)-VP1_(DSV)-VP2_(SV)-VP1VP10_(Houston/86)).

The fusions may comprise any number of VP1 and VP2 polypeptides fromdifferent isolates of Norovirus and/or any number of VP1 and VP10polypeptides from different isolates of Sapovirus, for example, fusionproteins may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 or more VP1, VP2, and/or VP10polypeptides, which may be present in any order in the multiepitopefusion protein. Fusion proteins may comprise the same or differentnumbers of VP1, VP2, and VP10 polypeptides.

In certain embodiments, the fusion proteins comprise one or moreORF1-encoded nonstructural polypeptides from one or more isolates ofNorovirus (e.g., Nterm, NTPase, p20, p22, VPg, Pro, and Pol) and/orSapovirus (e.g., p11, p28, NTPase, p32, VPg, Pro, Pol, and VP1). Fusionproteins may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 or more nonstructural polypeptides. Thesenonstructural polypeptides need not be in the order in which theynaturally occur in the native Norovirus or Sapovirus polyproteins. Thus,for example, an Nterm polypeptide may be at the N- and/or C-terminus ofa fusion protein. Multiple copies of a particular nonstructuralpolypeptide from different isolates of Norovirus and/or Sapovirus may bepresent in the fusion protein. In certain embodiments, the fusionproteins may further comprise one or more structural proteins (e.g.,VP1, VP2, and VP10) from one or more isolates of Norovirus and/orSapovirus.

In all fusions described herein, the viral regions need not be in theorder in which they occur naturally. Moreover, each of the regions canbe derived from the same or different Norovirus or Sapovirus isolates.The various Norovirus and Sapovirus polypeptides present in the variousfusions described above can either be full-length polypeptides orportions thereof.

In certain embodiments, the portions of the Norovirus and Sapoviruspolypeptides making up the fusion protein comprise at least one epitope,which is recognized by a T cell receptor on an activated T cell.Epitopes of VP1, VP2, VP10, Nterm, NTPase, p20, p22, VPg, Pro, Pol, p11,p28, p35, and p32 from Norovirus and Sapovirus isolates can beidentified by several methods. For example, the individual polypeptidesor fusion proteins comprising any combination of the above, can beisolated, by, e.g., immunoaffinity purification using a monoclonalantibody for the polypeptide or protein. The isolated protein sequencecan then be screened by preparing a series of short peptides byproteolytic cleavage of the purified protein, which together span theentire protein sequence. By starting with, for example, 100-merpolypeptides, each polypeptide can be tested for the presence ofepitopes recognized by a T-cell receptor on a Norovirus orSapovirus-activated T cell, progressively smaller and overlappingfragments can then be tested from an identified 100-mer to map theepitope of interest.

Epitopes recognized by a T-cell receptor on a Norovirus- orSapovirus-activated T cell can be identified by, for example, ⁵¹Crrelease assay (see Example 4) or by lymphoproliferation assay (seeExample 6). In a ⁵¹Cr release assay, target cells can be constructedthat display the epitope of interest by cloning a polynucleotideencoding the epitope into an expression vector and transforming theexpression vector into the target cells. Norovirus-specific orSapovirus-specific CD8⁺ T cells will lyse target cells displaying, forexample, one or more epitopes from one or more Norovirus or Sapoviruspolypeptides found in the fusion, and will not lyse cells that do notdisplay such an epitope. In a lymphoproliferation assay,Norovirus-activated and/or Sapovirus-activated CD4⁺ T cells willproliferate when cultured with, for example, one or more epitopes fromone or more Norovirus and/or Sapovirus polypeptides found in the fusion,but not in the absence of a Norovirus or Sapovirus epitopic peptide.

Useful polypeptides in the fusion include T-cell epitopes derived fromany of the various regions in polyproteins or structural proteins, VP1,VP2, and VP10. In this regard, Norovirus capsid proteins are known tocontain human T-cell epitopes (see, e.g., Nicollier-Jamot et al. (2004)Vaccine 22:1079-1086). Including one or more T-cell epitopes (both CD4+and CD8+) serves to increase vaccine efficacy as well as to increaseprotective levels against multiple Norovirus and/or Sapovirus genotypes.Moreover, multiple copies of specific, conserved T-cell epitopes canalso be used in the fusions, such as a composite of epitopes fromdifferent genotypes.

For example, polypeptides from the VP1 and VP2 regions can be used inthe fusions of the present invention. Immunogenic fragments of VP1and/or VP2 which comprise epitopes may be used in the subject fusions.For example, fragments of VP1 polypeptides can comprise from about 5 tonearly the full-length of the molecule, such as 6, 10, 25, 50, 75, 100,125, 150, 175, 200, 250, 300, 350, 400, 500 or more amino acids of a VP1polypeptide, or any integer between the stated numbers. Similarly,fragments of VP2 polypeptides can comprise 6, 10, 25, 50, 75, 100, 150,175, or 200 amino acids of a VP2 polypeptide, or any integer between thestated numbers.

If desired, the fusion proteins, or the individual components of theseproteins, also can contain other amino acid sequences, such as aminoacid linkers or signal sequences, as well as ligands useful in proteinpurification, such as glutathione-S-transferase and staphylococcalprotein A.

B. Nucleic Acids

Nucleic acids for use in the invention, for example, in polypeptideproduction, VLP production, and/or nucleic acid immunization, can bederived from any of the various regions of a Norovirus or Sapovirusgenome, including from any of the ORF1, ORF2, or ORF3 regions.Representative sequences from Norovirus and Sapovirus isolates arelisted herein. Thus, nucleic acids for use in the invention includethose derived from one or more sequences from any pathogenic Norovirusor Sapovirus genotype or isolate.

Representative sequences from Norovirus are known and are presented inFIGS. 1A-1C, 2A-2D, 14A-14B, and 15A-15B, and SEQ ID NOS:1-9 and SEQ IDNOS:13-17. Additional representative Norovirus sequences are Norwalkvirus, GenBank Accession No. M87661, Snow Mountain virus, GenBankAccession No. U70059; Snow Mountain virus, GenBank Accession No.AY134748, Hawaii virus; GenBank Accession No. U07611, and sequencesdisclosed in the following patent publications: WO 05/030806, WO00/79280, JP2002020399, US2003129588, U.S. Pat. No. 6,572,862, WO94/05700, and WO 05/032457. See also Green et al. (2000) J. Infect. Dis.181(Suppl. 2):S322-330; Wang et al. (0.1994) J. Virol. 68:5982-5990;Chen et al. (2004) J. Virol. 78: 6469-6479; Chakravarty et al. (2005) J.Virol. 79: 554-568; and Fankhauser et al. (1998) J. Infect. Dis.178:1571-1578; for sequence comparisons of different Norovirus strains.

Representative sequences from Sapovirus are also known and are presentedin SEQ ID NOS:10-12, 18, and 19. Additional representative Sapovirussequences are Sapporo virus-London/29845, GenBank Accession No. U95645,Parkville virus, GenBank Accession No. AF294739; and Sapporovirus-Houston/86, GenBank Accession No. U95643. See also Schuffeneckeret al. (2001) Arch Virol.; 146(11):2115-2132; Zintz et al. (2005)Infect. Genet. Evol. 5:281-290; Farkas et al. (2004) Arch. Virol.149:1309-1323; for sequence comparisons of different Sapovirus strains.

Any of these sequences, as well as fragments and variants thereof thatcan be used in nucleic acid immunization to elicit an immune response toa Norovirus or Sapovirus will find use in the present methods. Thus, theinvention includes variants of the above sequences displaying at leastabout 80-100% sequence identity thereto, including any percent identitywithin these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity thereto. Theinvention also includes polynucleotides encoding immunogenic fragmentsof a Norovirus or Sapovirus polypeptide derived from any of the abovesequences or a variant thereof. Polynucleotides can also comprise codingsequences for polypeptides which occur naturally or can be artificialsequences which do not occur in nature.

Polynucleotides may contain less than an entire Norovirus or Sapovirusgenome, or alternatively can include the sequence of an entire viralgenomic RNA. For example, polynucleotides may comprise one or moresequences from the ORF1, ORF2, and ORF3 regions of a Norovirus orSapovirus genome. Polynucleotides may also comprise the entire viralgenomic RNA or less than the entire viral genomic RNA from multiplegenotypes and/or isolates of Norovirus or Sapovirus.

In certain embodiments, polynucleotides comprise an ORF1 sequence codingfor the full-length polyprotein of a Norovirus or Sapovirus. In otherembodiments, polynucleotides comprise one or more portions of the ORF1sequence of a Norovirus or Sapovirus, for example, polynucleotides maycomprise sequences coding for one or more Norovirus ORF1-encodedpolypeptides, such as the N-terminal protein, NTPase, p20, VPg,protease, polymerase, VP1, and VP2, or one or more Sapoviruspolypeptides, such as the N-terminal protein, p11, p28, NTPase, p32;VPg, protease, polymerase, and VP1; or fragments thereof.

For example, a polynucleotide may comprise an ORF1 nucleotide sequenceselected from the group consisting of: a) a sequence comprisingcontiguous nucleotides 5-994 of ORF1, b) a sequence comprisingcontiguous nucleotides 995-2092 of ORF1, c) a sequence comprisingcontiguous nucleotides 2093-2629 of ORF1, d) a sequence comprisingcontiguous nucleotides 2630-3028 of ORF1, e) a sequence comprisingcontiguous nucleotides 3029-3271 of ORF1, and f) a sequence comprisingcontiguous nucleotides 3272-5101 of ORF1. The foregoing numbering isrelative to the ORF1 nucleotide sequence of Norovirus strain MD145-12(SEQ ID NO:13), and it is to be understood that the correspondingnucleotide positions in ORF1 sequences obtained from other genotypes andisolates of Norovirus and Sapovirus are also intended to be encompassedby the present invention.

In another example, a polynucleotide may comprise a nucleotide sequenceencoding a portion of a Norovirus or Sapovirus polyprotein. In certainembodiments, the polynucleotide is selected from the group consistingof: a) a polynucleotide encoding an amino acid sequence comprisingcontiguous amino acids 1-330 of an ORF1-encoded polyprotein, b) apolynucleotide encoding an amino acid sequence comprising contiguousamino acids 331-696 of an ORF1-encoded polyprotein, c) a polynucleotideencoding an amino acid sequence comprising contiguous amino acids697-875 of an ORF1-encoded polyprotein, d) a polynucleotide encoding anamino acid sequence comprising contiguous amino acids 876-1008 of anORF1-encoded polyprotein, e) a polynucleotide encoding an amino acidsequence comprising contiguous amino acids 1009-1189 of an ORF1-encodedpolyprotein, and f) a polynucleotide encoding an amino acid sequencecomprising contiguous amino acids 1090-1699 of an ORF1-encodedpolyprotein. The foregoing numbering is relative to the polyproteinamino acid sequence of Norovirus strain MD145-12 (SEQ ID NO:14), and itis to be understood that the corresponding amino acid positions inpolyprotein sequences obtained from other genotypes and isolates ofNorovirus and Sapovirus are also intended to be encompassed by thepresent invention.

In certain embodiments, the polynucleotides comprise sequences encodingone or more capsid proteins of a Norovirus or Sapovirus. For example,polynucleotides may comprise one or more sequences coding for structuralproteins (e.g., VP1, VP2, VP10) of a Norovirus or Sapovirus. In certainembodiments, the polynucleotide is selected from the group consistingof: a) a polynucleotide comprising contiguous nucleotides 5085-6701 of aNorovirus genomic nucleic acid numbered relative to Norovirus strainMD145-12 (SEQ ID NO:13), b) a polynucleotide comprising contiguousnucleotides 6704-7507 of a Norovirus genomic nucleic acid numberedrelative to Norovirus strain MD145-12 (SEQ ID NO:13), c) apolynucleotide comprising contiguous nucleotides 5174-6847 of aSapovirus genomic nucleic acid numbered relative to Sapovirus strainMc10 (SEQ ID NO:18), and d) a polynucleotide comprising contiguousnucleotides 6856-7350 of a Sapovirus genomic nucleic acid numberedrelative to Sapovirus strain Mc10 (SEQ ID NO:18). In certainembodiments, polynucleotides comprise sequences coding for at least twocapsid proteins from multiple genotypes and/or isolates of Norovirus andSapovirus.

In certain embodiments, polynucleotides comprise one or more NorovirusORF2 and ORF3 sequences from one or more isolates of Norovirus. In oneembodiment, polynucleotides comprise an ORF2 sequence coding for themajor capsid protein (VP1) of a Norovirus. In another embodiment,polynucleotides comprise an ORF3 sequence coding for the minorstructural protein (VP2) of a Norovirus. In yet another embodiment,polynucleotides comprise both a sequence coding for the major capsidprotein and a sequence coding for the minor structural protein of aNorovirus.

In certain embodiments, polynucleotides comprise one or more Sapovirussequences coding for the capsid proteins of one or more isolates ofSapovirus. In certain embodiments, polynucleotides comprise one or moresequences coding for the capsid proteins of one or more isolates ofSapovirus and one or more Norovirus ORF2 and/or ORF3 sequences of one ormore isolates of Norovirus.

In certain embodiments, the invention provides polynucleotides encodinga multiepitope fusion protein as described herein. Multiepitope fusionproteins can comprise sequences from one or more genotypes and/orisolates of Norovirus or Sapovirus. The polynucleotides may encodefusion antigens comprising ORF1-encoded, ORF2-encoded, and/orORF3-encoded polypeptides or fragments thereof, including, for example,sequences of Norovirus polypeptides, such as N-terminal protein, NTPase,p20, VPg, protease, polymerase, VP1, and VP2; and/or sequences ofSapovirus polypeptides, such as N-terminal protein, p11, p28, NTPase,p32, VPg, protease, polymerase, VP1, and VP10. The sequences may bederived from multiple genotypes and/or isolates of Norovirus andSapovirus. The polynucleotides may also encode fusion antigenscomprising sequences exogenous to the Noroviruses or Sapoviruses. Apolynucleotide encoding a fusion protein can be constructed frommultiple oligonucleotides comprising sequences encoding fragments of thefusion protein by ligating the oligonucleotides to form a codingsequence for the full-length fusion protein using standard molecularbiology techniques. See, e.g., U.S. Pat. No. 6,632,601 and U.S. Pat. No.6,630,298.

In certain embodiments, the polynucleotide encoding the multiepitopefusion protein comprises a Norovirus ORF2 sequence coding for the majorcapsid protein of a Norovirus and at least one other sequence coding fora capsid protein from a different isolate of Norovirus or Sapovirus. Incertain embodiments, the polynucleotide encoding the multiepitope fusionprotein comprises a Norovirus ORF2 sequence coding for the major capsidprotein of a Norovirus and at least one other sequence from a differentregion of the Norovirus genome, such as an ORF1 or ORF3 sequence fromthe same or a different isolate of Norovirus or Sapovirus. In certainembodiments, the polynucleotide encoding the multiepitope fusion proteincomprises one or more sequences from the ORF1 region of a Norovirus orSapovirus. For example, polynucleotides may comprise sequences codingfor one or more Norovirus ORF1-encoded polypeptides, such as theN-terminal protein, NTPase, p20, VPg, protease, polymerase, VP1, andVP2, or one or more Sapovirus polypeptides, such as the N-terminalprotein, p11, p28, NTPase, p32, VPg, protease, polymerase, and VP1; orfragments thereof. In certain embodiments, the polynucleotide encodingthe multiepitope fusion protein comprises one or more sequences from theORF1 region of a Norovirus or Sapovirus and one or more sequences fromthe ORF2 or ORF3 regions of the same or a different isolate of Norovirusor Sapovirus. Polynucleotides of the invention can also comprise othernucleotide sequences, such as sequences coding for linkers, signalsequences, or ligands useful in protein purification such asglutathione-S-transferase and staphylococcal protein A.

Nucleic acids according to the invention can be prepared in many ways(e.g. by chemical synthesis, from genomic or cDNA libraries, from theorganism itself, etc.) and can take various forms (e.g. single stranded,double stranded, vectors, probes, etc.). Preferably, nucleic acids areprepared in substantially pure form (i.e. substantially free from otherhost cell or non host cell nucleic acids).

For example, nucleic acids can be obtained by screening cDNA and/orgenomic libraries from cells infected with virus, or by deriving thegene from a vector known to include the same. For example,polynucleotides of interest can be isolated from a genomic libraryderived from viral RNA, present in, for example, stool or vomit samplesfrom an infected individual. Alternatively, Norovirus or Sapovirusnucleic acids can be isolated from infected humans or other mammals orfrom stool or vomit samples collected from infected individuals asdescribed in e.g., Estes et al. U.S. Pat. No. 6,942,86; Guntapong et al.(2004) Jpn J. Infect. Dis. 57:276-278; Harrington et al. (2004) J.Virol. 78:3035-3045; Fankhauser et al. (1998) J. Infect. Dis.178:1571-1578; and Dolin et al. (1971) J. Infect. Dis. 123:307-312.Viruses can be grown in LLC-PK cells in the presence of intestinal fluidcontaining bile acids (Chang et al. (2004.) Proc. Natl. Acad. Sci.U.S.A. 101:8733-8738). An amplification method such as PCR can be usedto amplify polynucleotides from either Norovirus or Sapovirus genomicRNA or cDNA encoding therefor. Alternatively, polynucleotides can besynthesized in the laboratory, for example, using an automaticsynthesizer. The nucleotide sequence can be designed with theappropriate codons for the particular amino acid sequence desired. Ingeneral, one will select preferred codons for the intended host in whichthe sequence will be expressed. The complete sequence of thepolynucleotide of interest can be assembled from overlappingoligonucleotides prepared by standard methods and assembled into acomplete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambairet al. (1984) Science 223:1299; Jay et al. (1984) J. Biol. Chem.259:6311; Stemmer et al. (1995) Gene 164:49-53. The polynucleotides canbe RNA or single- or double-stranded DNA. Preferably, thepolynucleotides are isolated free of other components, such as proteinsand lipids.

Thus, particular nucleotide sequences can be obtained from vectorsharboring the desired sequences or synthesized completely or in partusing various oligonucleotide synthesis techniques known in the art,such as site-directed mutagenesis and polymerase chain reaction (PCR)techniques where appropriate. See, e.g., Sambrook, supra. In particular,one method of obtaining nucleotide sequences encoding the desiredsequences is by annealing complementary sets of overlapping syntheticoligonucleotides produced in a conventional, automated polynucleotidesynthesizer, followed by ligation with an appropriate DNA ligase andamplification of the ligated nucleotide sequence via PCR. See, e.g.,Jayaraman et al. (1991) Proc. Natl. Acad. Sci. USA 88:4084-4088.Additionally, oligonucleotide directed synthesis (Jones et al. (1986)Nature 54:75-82), oligonucleotide directed mutagenesis of pre-existingnucleotide regions (Riechmann et al. (1988) Nature 332:323-327 andVerhoeyen et al. (1988) Science 239:1534-1536), and enzymatic filling-inof gapped oligonucleotides using T₄ DNA polymerase (Queen et al. (1989)Proc. Natl. Acad. Sci. USA 86:10029-10033) can be used to providemolecules having altered or enhanced antigen-binding capabilities,and/or reduced immunogenicity.

C. Production of Immunogenic Polypeptides

Polypeptides described herein can be prepared in any suitable manner(e.g. recombinant expression, purification from cell culture, chemicalsynthesis, etc.) and in various forms (e.g. native, fusions,non-glycosylated, lipidated, etc.). Such polypeptides includenaturally-occurring polypeptides, recombinantly produced polypeptides,synthetically produced polypeptides, or polypeptides produced by acombination of these methods. Means for preparing such polypeptides arewell understood in the art. Polypeptides are preferably prepared insubstantially pure form (i.e. substantially free from other host cell ornon host cell proteins).

Polypeptides can be conveniently synthesized chemically, by any ofseveral techniques that are known to those skilled in the peptide art.In general, these methods employ the sequential addition of one or moreamino acids to a growing peptide chain. Normally, either the amino orcarboxyl group of the first amino acid is protected by a suitableprotecting group. The protected or derivatized amino acid can then beeither attached to an inert solid support or utilized in solution byadding the next amino acid in the sequence having the complementary(amino or carboxyl) group suitably protected, under conditions thatallow for the formation of an amide linkage. The protecting group isthen removed from the newly added amino acid residue and the next aminoacid (suitably protected) is then added, and so forth. After the desiredamino acids have been linked in the proper sequence, any remainingprotecting groups (and any solid support, if solid phase synthesistechniques are used) are removed sequentially or concurrently, to renderthe final polypeptide. By simple modification of this general procedure,it is possible to add more than one amino acid at a time to a growingchain, for example, by coupling (under conditions which do not racemizechiral centers) a protected tripeptide with a properly protecteddipeptide to form, after deprotection, a pentapeptide. See, e.g., J. M.Stewart and J. D. Young, Solid Phase Peptide Synthesis (Pierce ChemicalCo., Rockford, Ill. 1984) and G. Barany and R. B. Merrifield, ThePeptides: Analysis, Synthesis, Biology, editors E. Gross and J.Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, forsolid phase peptide synthesis techniques; and M. Bodansky, Principles ofPeptide Synthesis, (Springer-Verlag, Berlin 1984) and E. Gross and J.Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, Vol. 1,for classical solution synthesis.

Typical protecting groups include t-butyloxycarbonyl (Boc),9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz);p-toluenesulfonyl (Tx); 2,4-dinitrophenyl; benzyl (Bzl);biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl,isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl,acetyl, o-nitrophenylsulfonyl and the like. Typical solid supports arecross-linked polymeric supports. These can include divinylbenzenecross-linked-styrene-based polymers, for example,divinylbenzene-hydroxymethylstyrene copolymers,divinylbenzene-chloromethylstyrene copolymers anddivinylbenzene-benzhydrylaminopolystyrene copolymers.

The polypeptides of the present invention can also be chemicallyprepared by other methods such as by the method of simultaneous multiplepeptide synthesis. See, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985)82:5131-5135; U.S. Pat. No. 4,631,211.

Alternatively, the above-described immunogenic polypeptides,polyproteins, and multiepitope fusion proteins can be producedrecombinantly. Once coding sequences for the desired proteins have beenisolated or synthesized, they can be cloned into any suitable vector orreplicon for expression. Numerous cloning vectors are known to those ofskill in the art, and the selection of an appropriate cloning vector isa matter of choice. A variety of bacterial, yeast, plant, mammalian andinsect expression systems are available in the art and any suchexpression system can be used (e.g., see Examples 1 and 2 forconstruction of exemplary expression cassettes for expression in yeastand insect cells, respectively). Optionally, a polynucleotide encodingthese proteins can be translated in a cell-free translation system. Suchmethods are well known in the art.

Examples of recombinant DNA vectors for cloning and host cells whichthey can transform include the bacteriophage λ (E. coli), pBR322 (E.coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106(gram-negative bacteria), pLAFR1 (gram-negative bacteria), pME290(non-E. coli gram-negative bacteria), pHV14 (E. coli and Bacillussubtilis), pBD9 (Bacillus), 0.161 (Streptomyces), pUC6 (Streptomyces),YIp5 (Saccharomyces), YCp19 (Saccharomyces) and bovine papilloma virus(mammalian cells). See, generally, DNA Cloning: Vols. I & II, supra;Sambrook et al., supra; B. Perbal, supra.

Insect cell expression systems, such as baculovirus systems, can also beused and are known to those of skill in the art and described in, e.g.,Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987). Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, interalfa, Invitrogen, San Diego Calif. (“MaxBac” kit).

Plant expression systems can also be used to produce the immunogenicproteins. Generally, such systems use virus-based vectors to transfectplant cells with, heterologous genes. For a description of such systemssee, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221; and Hackiand etal., Arch. Virol. (1994) 139:1-22.

Viral systems, such as a vaccinia based infection/transfection system,as described in Tomei et al., J. Virol. (1993) 67:4017-4026 and Selby etal., J. Gen. Virol. (1993) 74:1103-1113, will also find use with thepresent invention: In this system, cells are first transfected in vitrowith a vaccinia virus recombinant that encodes the bacteriophage T7 RNApolymerase. This polymerase displays exquisite specificity in that itonly transcribes templates bearing T7 promoters. Following infection,cells are transfected with the DNA of interest, driven by a T7 promoter.The polymerase expressed in the cytoplasm from the vaccinia virusrecombinant transcribes the transfected DNA into RNA which is thentranslated into protein by the host translational machinery. The methodprovides for high level, transient, cytoplasmic production of largequantities of RNA and its translation product(s).

The gene can be placed under the control of a promoter, ribosome bindingsite (for bacterial expression) and, optionally, an operator(collectively referred to herein as “control” elements), so that the DNAsequence encoding the desired immunogenic polypeptide is transcribedinto RNA in the host cell transformed by a vector containing thisexpression construction. The coding sequence may or may not contain asignal peptide or leader sequence. With the present invention, both thenaturally occurring signal peptides or heterologous sequences can beused. Leader sequences can be removed by the host in post-translationalprocessing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397.Such sequences include, but are not limited to, the tpa leader, as wellas the honey bee mellitin signal sequence.

Other regulatory sequences may also be desirable which allow forregulation of expression of the protein sequences relative to the growthof the host cell. Such regulatory sequences are known to those of skillin the art, and examples include those which cause the expression of agene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Other typesof regulatory elements may also be present in the vector, for example,enhancer sequences.

The control sequences and other regulatory sequences may be ligated tothe coding sequence prior to insertion into a vector. Alternatively, thecoding sequence can be cloned directly into an expression vector whichalready contains the control sequences and an appropriate restrictionsite.

In some cases it may be necessary to modify the coding sequence so thatit may be attached to the control sequences with the appropriateorientation; i.e., to maintain the proper reading frame. It may also bedesirable to produce mutants or analogs of the immunogenic polypeptides.Mutants or analogs may be prepared by the deletion of a portion of thesequence encoding the protein, by insertion of a sequence, and/or bysubstitution of one or more nucleotides within the sequence. Techniquesfor modifying nucleotide sequences, such as site-directed mutagenesis,are well known to those skilled in the art. See, e.g., Sambrook et al.,supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization,supra.

The expression vector is then used to transform an appropriate hostcell. A number of mammalian cell lines are known in the art and includeimmortalized cell lines available from the American Type CultureCollection (ATCC), such as, but not limited to, Chinese hamster ovary(CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidneycells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), aswell as others. Similarly, bacterial hosts such as E. coli, Bacillussubtilis, and Streptococcus spp., will find use with the presentexpression constructs. Yeast hosts useful in the present inventioninclude inter alia, Saccharomyces cerevisiae, Candida albicans, Candidamaltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyceslactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomycespombe and Yarrowia lipolytica. Insect cells for use with baculovirusexpression vectors include, inter alia, Aedes aegypti, Autographacalifornica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni.

Depending on the expression system and host selected, the proteins ofthe present invention are produced by growing host cells transformed byan expression vector described above under conditions whereby theprotein of interest is expressed. The selection of the appropriategrowth conditions is within the skill of the art. The cells are thendisrupted, using chemical, physical or mechanical means, which lyse thecells yet keep the Norovirus and/or Sapovirus immunogenic polypeptidessubstantially intact. Intracellular proteins can also be obtained byremoving components from the cell wall or membrane, e.g., by the use ofdetergents or organic solvents, such that leakage of the immunogenicpolypeptides occurs. Such methods are known to those of skill in the artand are described in, e.g., Protein Purification Applications: APractical Approach, (E. L. V. Harris and S. Angal, Eds., 1990).

For example, methods of disrupting cells for use with the presentinvention include but are not limited to: sonication or ultrasonication;agitation; liquid or solid extrusion; heat treatment; freeze-thaw;desiccation; explosive decompression; osmotic shock; treatment withlytic enzymes including proteases such as trypsin, neuraminidase andlysozyme; alkali treatment; and the use of detergents and solvents suchas bile salts, sodium dodecylsulphate, Triton, NP40 and CHAPS. Theparticular technique used to disrupt the cells is largely a matter ofchoice and will depend on the cell type in which the polypeptide isexpressed, culture conditions and any pre-treatment used.

Following disruption of the cells, cellular debris is removed, generallyby centrifugation, and the intracellularly produced Norovirus and/orSapovirus immunogenic polypeptides are further purified, using standardpurification techniques such as but not limited to, columnchromatography, ion-exchange chromatography, size-exclusionchromatography, electrophoresis, HPLC, immunoabsorbent techniques,affinity chromatography, immunoprecipitation, and the like.

For example, one method for obtaining the intracellular Norovirus and/orSapovirus immunogenic polypeptides of the present invention involvesaffinity purification, such as by immunoaffinity chromatography usingspecific antibodies. The choice of a suitable affinity resin is withinthe skill in the art. After affinity purification, immunogenicpolypeptides can be further purified using conventional techniques wellknown in the art, such as by any of the techniques described above.

It may be desirable to produce multiple polypeptides simultaneously(e.g., structural and/or nonstructural proteins from one or more viralstrains or viral polypeptides in combination with polypeptideadjuvants). Production of two or more different polypeptides can readilybe accomplished by e.g., co-transfecting host cells with constructsencoding the different polypeptides. Co-transfection can be accomplishedeither in trans or cis, i.e., by using separate vectors or by using asingle vector encoding the polypeptides. If a single vector is used,expression of the polypeptides can be driven by a single set of controlelements or, alternatively, the sequences coding for the polypeptidescan be present on the vector in individual expression cassettes,regulated by individual control elements.

The polypeptides described herein may be attached to a solid support.The solid supports which can be used in the practice of the inventioninclude substrates such as nitrocellulose (e.g., in membrane ormicrotiter well form); polyvinylchloride (e.g., sheets or microtiterwells); polystyrene latex (e.g., beads or microtiter plates);polyvinylidine fluoride; diazotized paper; nylon membranes; activatedbeads, magnetically responsive beads, and the like.

Typically, a solid support is first reacted with a solid phase component(e.g., one or more Norovirus or Sapovirus antigens) under suitablebinding conditions such that the component is sufficiently immobilizedto the support. Sometimes, immobilization of the antigen to the supportcan be enhanced by first coupling the antigen to a protein with betterbinding properties. Suitable coupling proteins include, but are notlimited to, macromolecules such as serum albumins including bovine serumalbumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, and other proteins well known to those skilledin the art. Other molecules that can be used to bind the antigens to thesupport include polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and the like. Suchmolecules and methods of coupling these molecules to the antigens, arewell known to those of ordinary skill in the art. See, e.g., Brinkley,M. A., Bioconjugate Chem. (1992) 3:2-13; Hashida et al., J. Appl.Biochem. (1984) 6:56-63; and Anjaneyulu and Staros, International J. ofPeptide and Protein Res. (1987) 30:117-124.

If desired, polypeptides may be labeled using conventional techniques.Suitable labels include fluorophores, chromophores, radioactive atoms(particularly ³²P and ¹²⁵I, electron-dense reagents, enzymes, andligands having specific binding partners. Enzymes are typically detectedby their activity. For example, horseradish peroxidase is usuallydetected by its ability to convert 3,3′,5,5′-tetramethylbenzidine (TMB)to a blue pigment, quantifiable with a spectrophotometer. “Specificbinding partner” refers to a protein capable of binding a ligandmolecule with high specificity, as for example in the case of an antigenand a monoclonal antibody specific therefor. Other specific bindingpartners include biotin and avidin or streptavidin, IgG and protein A,and the numerous receptor-ligand couples known in the art. A singlelabel or a combination of labels may be used in the practice of theinvention.

D. Nucleic Acid Immunization

Nucleic acid immunization using nucleic acids, described herein,encoding immunogenic capsid polypeptides and/or other immunogenic viralpolypeptides (e.g., structural and nonstructural proteins), and/ormultiepitope fusion proteins, and/or VLPs can be used to elicit animmune response in a subject, for example, to treat or prevent Norovirusand/or Sapovirus infection.

Nucleic acids described herein can be inserted into an expression vectorto create an expression cassette capable of producing the viralpolypeptides and/or VLPs in a suitable host cell. The ability ofVP1-encoding constructs to produce VLPs can be empirically determined(e.g., see Examples 1 and 2 describing detection of VLPs by electronmicroscopy).

Expression cassettes typically include control elements operably linkedto the coding sequence, which allow for the expression of the gene invivo in the subject species. For example, typical promoters formammalian cell expression include the SV40 early promoter, a CMVpromoter such as the CMV immediate early promoter, the mouse mammarytumor virus LTR promoter, the adenovirus major late promoter (Ad MLP),and the herpes simplex virus promoter, among others. Other nonviralpromoters, such as a promoter derived from the murine metallothioneingene, will also find use for mammalian expression. Typically,transcription termination and polyadenylation sequences will also bepresent, located 3′ to the translation stop codon. Preferably, asequence for optimization of initiation of translation, located 5′ tothe coding sequence, is also present. Examples of transcriptionterminator/polyadenylation signals include those derived from SV40, asdescribed in Sambrook et al., supra, as well as a bovine growth hormoneterminator sequence.

Enhancer elements may also be used herein to increase expression levelsof the mammalian constructs. Examples include the SV40 early geneenhancer, as described in Dijkema et al., EMPO J. (1985) 4:761, theenhancer/promoter derived from the long terminal repeat (LTR) of theRous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad.Sci. USA (1982b) 79:6777 and elements derived from human CMV, asdescribed in Boshart et al., Cell (1985) 41:521, such as elementsincluded in the CMV intron A sequence.

In addition, vectors can be constructed that include sequences codingfor adjuvants. Particularly suitable are detoxified mutants of bacterialADP-ribosylating toxins, for example, diphtheria toxin, pertussis toxin(PT), cholera toxin (CT), E. coli heat-labile toxins (LT1 and LT2),Pseudomonas endotoxin A, C. botulinum C2 and C3 toxins, as well astoxins from C. perfringens, C. spiriforma and C. dfficile. In apreferred embodiment, vectors include coding sequences for detoxifiedmutants of E. coli heat-labile toxins, such as the LT-K63 and LT-R72detoxified mutants, described in U.S. Pat. No. 6,818,222, hereinincorporated by reference in its entirety. One or more adjuvantpolypeptides may be coexpressed with Norovirus and/or Sapoviruspolypeptides. In certain embodiments, adjuvant and viral polypeptidesmay be coexpressed in the form of a fusion protein comprising one ormore adjuvant polypeptides and one or more viral polypeptides.Alternatively, adjuvant and viral polypeptides may be coexpressed asseparate proteins.

Furthermore, vectors can be constructed that include chimericantigen-coding gene sequences, encoding, e.g., multipleantigens/epitopes of interest, for example derived from a single or frommore than one viral isolate. In certain embodiments, adjuvant or antigencoding sequences precede or follow viral capsid coding sequences, andthe chimeric transcription unit has a single open reading frame encodingthe adjuvant and/or antigen of interest and the capsid polypeptide.Alternatively, multi-cistronic cassettes (e.g., bi-cistronic cassettes)can be constructed allowing expression of multiple adjuvants and/orantigens from a single mRNA using the EMCV IRES, or the like. Lastly,adjuvants and/or antigens can be encoded on separate transcripts fromindependent promoters on a single plasmid or other vector.

Once complete, the constructs are used for nucleic acid immunization orthe like using standard gene delivery protocols. Methods for genedelivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346,5,580,859, 5,589,466. Genes can be delivered either directly to thevertebrate subject or, alternatively, delivered ex vivo, to cellsderived from the subject and the cells reimplanted in the subject.

A number of viral based systems have been developed for gene transferinto mammalian cells. For example, retroviruses provide a convenientplatform for gene delivery systems. Selected sequences can be insertedinto a vector and packaged in retroviral particles using techniquesknown in the art. The recombinant virus can then be isolated anddelivered to cells of the subject either in vivo or ex vivo. A number ofretroviral systems have been described (U.S. Pat. No. 5,219,740; Millerand Rosman, BioTechniques (1989) 7:980-990; Miller, A. D., Human GeneTherapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burnset al., Proc. Natl. Acad. Sci. USA (1993) 90:8033-8037; and Boris-Lawrieand Temin, Cur. Opin. Genet. Develop. (1993) 3:102-109).

A number of adenovirus vectors have also been described. Unlikeretroviruses which integrate into the host genome, adenoviruses persistextrachromosomally thus minimizing the risks associated with insertionalmutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett etal., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human GeneTherapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barret al., Gene Therapy (1994) 1:51-58; Berkner, K. L. BioTechniques (1988)6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476).Additionally, various adeno-associated virus (AAV) vector systems havebeen developed for gene delivery. AAV vectors can be readily constructedusing techniques well known in the art. See, e.g., U.S. Pat. Nos.5,173,414 and 5,139,941; International Publication Nos. WO 92/01070(published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993);Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al.,Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J.Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. CurrentTopics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R. M. HumanGene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994)1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Another vector system useful for delivering the polynucleotides of thepresent invention is the enterically administered recombinant poxvirusvaccines described by Small, Jr., P. A., et al. (U.S. Pat. No.5,676,950, issued Oct. 14, 1997, herein incorporated by reference).

Additional viral vectors which will find use for delivering the nucleicacid molecules encoding the antigens of interest include those derivedfrom the pox family of viruses, including vaccinia virus and avianpoxvirus. By way of example, vaccinia virus recombinants expressing theNorovirus and/or Sapovirus antigens can be constructed as follows. TheDNA encoding the particular Norovirus or Sapovirus antigen codingsequence is first inserted into an appropriate vector so that it isadjacent to a vaccinia promoter and flanking vaccinia DNA sequences,such as the sequence encoding thymidine kinase (TK). This vector is thenused to transfect cells which are simultaneously infected with vaccinia.Homologous recombination serves to insert the vaccinia promoter plus thegene encoding the coding sequences of interest into the viral genome.The resulting TK-recombinant can be selected by culturing the cells inthe presence of 5-bromodeoxyuridine and picking viral plaques resistantthereto.

Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses,can also be used to deliver the genes. Recombinant avipox viruses,expressing immunogens from mammalian pathogens, are known to conferprotective immunity when administered to non-avian species. The use ofan avipox vector is particularly desirable in human and other mammalianspecies since members of the avipox genus can only productivelyreplicate in susceptible avian species and therefore are not infectivein mammalian cells. Methods for producing recombinant avipoxviruses areknown in the art and employ genetic recombination, as described abovewith respect to the production of vaccinia viruses. See, e.g., WO91/12882; WO 89/03429; and WO 92/03545.

Molecular conjugate vectors, such as the adenovirus chimeric vectorsdescribed in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 andWagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can alsobe used for gene delivery.

Members of the Alphavirus genus, such as, but not limited to, vectorsderived from the Sindbis virus (SIN), Semliki Forest virus (SFV), andVenezuelan Equine Encephalitis virus (VEE), will also find use as viralvectors for delivering the polynucleotides of the present invention. Fora description of Sindbis-virus derived vectors useful for the practiceof the instant methods, see, Dubensky et al. (1996) J. Virol.70:508-519; and International Publication Nos. WO 95/07995, WO 96/17072;as well as, Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723,issued Dec. 1, 1998, and Dubensky, Jr., T. W., U.S. Pat. No. 5,789,245,issued Aug. 4, 1998, both herein incorporated by reference. Particularlypreferred are chimeric alphavirus vectors comprised of sequences derivedfrom Sindbis virus and Venezuelan equine encephalitis virus. See, e.g.,Perri et al. (2003) J. Virol. 77: 10394-10403 and InternationalPublication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO00/61772; herein incorporated by reference in their entireties.

A vaccinia based infection/transfection system can be conveniently usedto provide for inducible, transient expression of the coding sequencesof interest (for example, a VP1/VP2 expression cassette) in a host cell.In this system, cells are first infected in vitro with a vaccinia virusrecombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the polynucleotide of interest, driven by a T7promoter. The polymerase expressed in the cytoplasm from the vacciniavirus recombinant transcribes the transfected DNA into RNA which is thentranslated into protein by the host translational machinery. The methodprovides for high level, transient, cytoplasmic production of largequantities of RNA and its translation products. See, e.g., Elroy-Steinand Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al.,Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

As an alternative approach to infection with vaccinia or avipox virusrecombinants, or to the delivery of genes using other viral vectors, anamplification system can be used that will lead to high level expressionfollowing introduction into host cells. Specifically, a T7 RNApolymerase promoter preceding the coding region for T7 RNA polymerasecan be engineered. Translation of RNA derived from this template willgenerate T7 RNA polymerase which in turn will transcribe more template.Concomitantly, there will be a cDNA whose expression is under thecontrol of the T7 promoter. Thus, some of the T7 RNA polymerasegenerated from translation of the amplification template RNA will leadto transcription of the desired gene. Because some T7 RNA polymerase isrequired to initiate the amplification, T7 RNA polymerase can beintroduced into cells along with the template(s) to prime thetranscription reaction. The polymerase can be introduced as a protein oron a plasmid encoding the RNA polymerase. For a further discussion of T7systems and their use for transforming cells, see, e.g., InternationalPublication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986)189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al.,Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc.Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994)22:2114-2120; and U.S. Pat. No. 5,135,855.

The synthetic expression cassette of interest can also be deliveredwithout a viral vector. For example, the synthetic expression cassettecan be packaged as DNA or RNA in liposomes prior to delivery to thesubject or to cells derived therefrom. Lipid encapsulation is generallyaccomplished using liposomes which are able to stably bind or entrap andretain nucleic acid. The ratio of condensed DNA to lipid preparation canvary but will generally be around 1:1 (mg DNA:micromoles lipid), or moreof lipid. For a review of the use of liposomes as carriers for deliveryof nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991)1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol.101, pp. 512-527.

Liposomal preparations for use in the present invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations, with cationic liposomes particularly preferred. Cationicliposomes have been shown to mediate intracellular delivery of plasmidDNA (Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416);mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081);and purified transcription factors (Debs et al., J. Biol. Chem. (1990)265:10189-10192), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Feigner et al., Proc. Natl. Acad. Sci. USA (1987)84:7413-7416). Other commercially available lipids include (DDAB/DOPE)and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be preparedfrom readily available materials using techniques well known in the art.See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198;PCT Publication No. WO 90/11092 for a description of the synthesis ofDOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such as,from Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). Thevarious liposome-nucleic acid complexes are prepared using methods knownin the art. See, e.g., Straubinger et al., in METHODS OF IMMUNOLOGY(1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA(1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta(1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham,Biochim Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA(1979) 76:145); Fraley et al., J. Biol. Chem. (1980) 255:10431; Szokaand Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; andSchaefer-Ridder et al., Science (1982) 215:166.

The DNA and/or protein antigen(s) can also be delivered in cochleatelipid compositions similar to those described by Papahadjopoulos et al.,Biochem. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Pat. Nos.4,663,161 and 4,871,488.

The expression cassette of interest may also be encapsulated, adsorbedto, or associated with, particulate carriers. Such carriers presentmultiple copies of a selected antigen to the immune system and promotemigration, trapping and retention of antigens in local lymph nodes. Theparticles can be taken up by profession antigen presenting cells such asmacrophages and dendritic cells, and/or can enhance antigen presentationthrough other mechanisms such as stimulation of cytokine release.Examples of particulate carriers include those derived from polymethylmethacrylate polymers, as well as microparticles derived frompoly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g.,Jeffery et al., Pharm. Res. (1993) 10:362-368; McGee J. P., et al., J.Microencapsul. 14(2):197-210, 1997; O'Hagan D. T., et al., Vaccine11(2):149-54, 1993.

Furthermore, other particulate systems and polymers can be used for thein vivo or ex vivo delivery of the gene of interest. For example,polymers such as polylysine, polyarginine, polyornithine, spermine,spermidine, as well as conjugates of these molecules, are useful fortransferring a nucleic acid of interest. Similarly, DEAEdextran-mediated transfection, calcium phosphate precipitation orprecipitation using other insoluble inorganic salts, such as strontiumphosphate, aluminum silicates including bentonite and kaolin, chromicoxide, magnesium silicate, talc, and the like, will find use with thepresent methods. See, e.g., Feigner, P. L., Advanced Drug DeliveryReviews (1990) 5:163-187, for a review of delivery systems useful forgene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No.5,831,005, issued Nov. 3, 1998, herein incorporated by reference) mayalso be used for delivery of a construct of the present invention.

Additionally, biolistic delivery systems employing particulate carrierssuch as gold and tungsten, are especially useful for deliveringsynthetic expression cassettes of the present invention. The particlesare coated with the synthetic expression cassette(s) to be delivered andaccelerated to high velocity, generally under a reduced atmosphere,using a gun powder discharge from a “gene gun.” For a description ofsuch techniques, and apparatuses useful therefore, see, e.g., U.S. Pat.Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and5,478,744. Also, needle-less injection systems can be used (Davis, H.L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

Recombinant vectors carrying a synthetic expression cassette of thepresent invention are formulated into compositions for delivery to avertebrate subject. These compositions may either be prophylactic (toprevent infection) or therapeutic (to treat disease after infection).The compositions will comprise a “therapeutically effective amount” ofthe gene of interest such that an amount of the antigen can be producedin vivo so that an immune response is generated in the individual towhich it is administered. The exact amount necessary will vary dependingon the subject being treated; the age and general condition of thesubject to be treated; the capacity of the subject's immune system tosynthesize antibodies; the degree of protection desired; the severity ofthe condition being treated; the particular antigen selected and itsmode of administration, among other factors. An appropriate effectiveamount can be readily determined by one of skill in the art. Thus, a“therapeutically effective amount” will fall in a relatively broad rangethat can be determined through routine trials.

The compositions will generally include one or more “pharmaceuticallyacceptable excipients or vehicles” such as water, saline, glycerol,polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally,auxiliary substances, such as wetting or emulsifying agents, pHbuffering substances, surfactants and the like, may be present in suchvehicles. Certain facilitators of immunogenicity or of nucleic aciduptake and/or expression can also be included in the compositions orcoadministered, such as, but not limited to, bupivacaine, cardiotoxinand sucrose.

Once formulated, the compositions of the invention can be administereddirectly to the subject (e.g., as described above) or, alternatively,delivered ex vivo, to cells derived from the subject, using methods suchas those described above. For example, methods for the ex vivo deliveryand reimplantation of transformed cells into a subject are known in theart and can include, e.g., dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, lipofectamineand LT-1 mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) (with or without thecorresponding antigen) in liposomes, and direct microinjection of theDNA into nuclei.

Direct delivery of synthetic expression cassette compositions in vivowill generally be accomplished with or without viral vectors, asdescribed above, by injection using either a conventional syringe,needless devices such as Bioject™ or a gene gun, such as the Accell™gene delivery system (PowderMed Ltd, Oxford, England). The constructscan be delivered (e.g., injected) either subcutaneously, epidermally,intradermally, intramuscularly, intravenous, intramucosally (such asnasally, rectally and vaginally), intraperitoneally or orally. Deliveryof DNA into cells of the epidermis is particularly preferred as thismode of administration provides access to skin-associated lymphoid cellsand provides for a transient presence of DNA in the recipient. Othermodes of administration include oral ingestion and pulmonaryadministration, suppositories, needle-less injection, transcutaneous,topical, and transdermal applications. Dosage treatment may be a singledose schedule or a multiple dose schedule.

Ex Vivo Delivery

In one embodiment, T cells, and related cell types (including but notlimited to antigen presenting cells, such as, macrophage, monocytes,lymphoid cells, dendritic cells, B-cells, T-cells, stem cells, andprogenitor cells thereof), can be used for ex vivo delivery ofexpression cassettes of the present invention. T cells can be isolatedfrom peripheral blood lymphocytes (PBLs) by a variety of proceduresknown to those skilled in the art. For example, T cell populations canbe “enriched” from a population of PBLs through the removal of accessoryand B cells. In particular, T cell enrichment can be accomplished by theelimination of non-T cells using anti-MHC class II monoclonalantibodies. Similarly, other antibodies can be used to deplete specificpopulations of non-T cells. For example, anti-Ig antibody molecules canbe used to deplete B cells and anti-MacI antibody molecules can be usedto deplete macrophages.

T cells can be further fractionated into a number of differentsubpopulations by techniques known to those skilled in the art. Twomajor subpopulations can be isolated based on their differentialexpression of the cell surface markers CD4 and CD8. For example,following the enrichment of T cells as described above, CD4⁺ cells canbe enriched using antibodies specific for CD4 (see Coligan et al.,supra). The antibodies may be coupled to a solid support such asmagnetic beads. Conversely, CD8+ cells can be enriched through the useof antibodies specific for CD4 (to remove CD4⁺ cells), or can beisolated by the use of CD8 antibodies coupled to a solid support. CD4lymphocytes from Norovirus or Sapovirus infected patients can beexpanded ex vivo, before or after transduction as described by Wilsonet. al. (1995) J. Infect. Dis. 172:88.

Following purification of T cells, a variety of methods of geneticmodification known to those skilled in the art can be performed usingnon-viral or viral-based gene transfer vectors constructed as describedherein. For example, one such approach involves transduction of thepurified T cell population with vector-containing supernatant ofcultures derived from vector producing cells. A second approach involvesco-cultivation of an irradiated monolayer of vector-producing cells withthe purified T cells. A third approach involves a similar co-cultivationapproach; however, the purified T cells are pre-stimulated with variouscytokines and cultured 48 hours prior to the co-cultivation with theirradiated vector producing cells. Pre-stimulation prior to suchtransduction increases effective gene transfer (Nolta et al. (1992) Exp.Hematol. 20:1065). Stimulation of these cultures to proliferate alsoprovides increased cell populations for re-infusion into the patient.Subsequent to co-cultivation, T cells are collected from the vectorproducing cell monolayer, expanded, and frozen in liquid nitrogen.

Gene transfer vectors, containing one or more expression cassettes ofthe present invention (associated with appropriate control elements fordelivery to the isolated T cells) can be assembled using known methods.

Selectable markers can also be used in the construction of gene transfervectors. For example, a marker can be used which imparts to a mammaliancell transduced with the gene transfer vector resistance to a cytotoxicagent. The cytotoxic agent can be, but is not limited to, neomycin,aminoglycoside, tetracycline, chloramphenicol, sulfonamide, actinomycin,netropsin, distamycin A, anthracycline, or pyrazinamide. For example,neomycin phosphotransferase II imparts resistance to the neomycinanalogue geneticin (G418).

The T cells can also be maintained in a medium containing at least onetype of growth factor prior to being selected. A variety of growthfactors are known in the art which sustain the growth of a particularcell type. Examples of such growth factors are cytokine mitogens such asrIL-2, IL-10, IL-12, and IL-15, which promote growth and activation oflymphocytes. Certain types of cells are stimulated by other growthfactors such as hormones, including human chorionic gonadotropin (hCG)and human growth hormone. The selection of an appropriate growth factorfor a particular cell population is readily accomplished by one of skillin the art.

For example, white blood cells such as differentiated progenitor andstem cells are stimulated by a variety of growth factors. Moreparticularly, IL-3, IL-4, IL-5, IL-6, IL-9, GM-CSF, M-CSF, and G-CSF,produced by activated T_(H) and activated macrophages, stimulate myeloidstem cells, which then differentiate into pluripotent stem cells,granulocyte-monocyte progenitors, eosinophil progenitors, basophilprogenitors, megakaryocytes, and erythroid progenitors. Differentiationis modulated by growth factors such as GM-CSF, IL-3, IL-6, IL-11, andEPO.

Pluripotent stem cells then differentiate into lymphoid stem cells, bonemarrow stromal cells, T cell progenitors, B cell progenitors,thymocytes, T_(H) cells, T_(c) cells, and B cells. This differentiationis modulated by growth factors such as IL-3, IL-4, IL-6, IL-7, GM-CSF,M-CSF, G-CSF, IL-2, and IL-5.

Granulocyte-monocyte progenitors differentiate to monocytes,macrophages, and neutrophils. Such differentiation is modulated by thegrowth factors GM-CSF, M-CSF, and IL-8. Eosinophil progenitorsdifferentiate into eosinophils. This process is modulated by GM-CSF andIL-5.

The differentiation of basophil progenitors into mast cells andbasophils is modulated by GM-CSF, IL-4, and IL-9. Megakaryocytes produceplatelets in response to GM-CSF, EPO, and IL-6. Erythroid progenitorcells differentiate into red blood cells in response to EPO.

Thus, during activation by the CD3-binding agent, T cells can also becontacted with a mitogen, for example a cytokine such as IL-2. Inparticularly preferred embodiments, IL-2 is added to the population of Tcells at a concentration of about 50 to 100 μg/ml. Activation with theCD3-binding agent can be carried out for 2 to 4 days.

Once suitably activated, the T cells are genetically modified bycontacting the same with a suitable gene transfer vector underconditions that allow for transfection of the vectors into the T cells.Genetic modification is carried out when the cell density of the T cellpopulation is between about 0.1×10⁶ and 5×10⁶, preferably between about0.5×10⁶ and 2×10⁶. A number of suitable viral and nonviral-based genetransfer vectors have been described for use herein.

After transduction, transduced cells are selected away fromnon-transduced cells using known techniques. For example, if the genetransfer vector used in the transduction includes a selectable markerwhich confers resistance to a cytotoxic agent, the cells can becontacted with the appropriate cytotoxic agent, whereby non-transducedcells can be negatively selected away from the transduced cells. If theselectable marker is a cell surface marker, the cells can be contactedwith a binding agent specific for the particular cell surface marker,whereby the transduced cells can be positively selected away from thepopulation. The selection step can also entail fluorescence-activatedcell sorting (FACS) techniques, such as where FACS is used to selectcells from the population containing a particular surface marker, or theselection step can entail the use of magnetically responsive particlesas retrievable supports for target cell capture and/or backgroundremoval.

More particularly, positive selection of the transduced cells can beperformed using a FACS cell sorter (e.g. a FACSVantage™ Cell Sorter,Becton Dickinson Immunocytometry Systems, San Jose, Calif.) to sort andcollect transduced cells expressing a selectable cell surface marker.Following transduction, the cells are stained with fluorescent-labeledantibody molecules directed against the particular cell surface marker.The amount of bound antibody on each cell can be measured by passingdroplets containing the cells through the cell sorter. By imparting anelectromagnetic charge to droplets containing the stained cells, thetransduced cells can be separated from other cells. The positivelyselected cells are then harvested in sterile collection vessels. Thesecell sorting procedures are described in detail, for example, in theFACSVantage™ Training Manual, with particular reference to sections 3-11to 3-28 and 10-1 to 10-17.

Positive selection of the transduced cells can also be performed usingmagnetic separation of cells based on expression or a particular cellsurface marker. In such separation techniques, cells to be positivelyselected are first contacted with specific binding agent (e.g., anantibody or reagent the interacts specifically with the cell surfacemarker). The cells are then contacted with retrievable particles (e.g.,magnetically responsive particles) which are coupled with a reagent thatbinds the specific binding agent (that has bound to the positive cells).The cell-binding agent-particle complex can then be physically separatedfrom non-labeled cells, for example using a magnetic field. When usingmagnetically responsive particles, the labeled cells can be retained ina container using a magnetic filed while the negative cells are removed.These and similar separation procedures are known to those of ordinaryskill in the art.

Expression of the vector in the selected transduced cells can beassessed by a number of assays known to those skilled in the art. Forexample, Western blot or Northern analysis can be employed depending onthe nature of the inserted nucleotide sequence of interest. Onceexpression has been established and the transformed T cells have beentested for the presence of the selected synthetic expression cassette,they are ready for infusion into a patient via the peripheral bloodstream. The invention includes a kit for genetic modification of an exvivo population of primary mammalian cells. The kit typically contains agene transfer vector coding for at least one selectable marker and atleast one synthetic expression cassette contained in one or morecontainers, ancillary reagents or hardware, and instructions for use ofthe kit.

E. Production of Viral-Like Particles

The capsid proteins of Noroviruses and Sapoviruses self-assemble intononinfectious virus-like particles (VLP) when expressed in variouseucaryotic cells (Taube et al. (2005) Arch Virol. 150:1425-1431; Ball etal. (1998) J. Virol. 72:1345-1353; Green et al. (1997) J. Clin.Microbiol. 35:1909-1914; Huang et al. (2005) Vaccine 23:1851-1858;Hansman et al. (2005) Arch. Virol. 150:21-36; herein incorporated byreference in their entireties). VLPs spontaneously form when aparticle-forming polypeptide of interest, for example, a Norovirus orSapovirus VP1 polypeptide or a variant or fragment thereof capable ofproducing VLPs, is recombinantly expressed in an appropriate host cell.

Expression vectors comprising Norovirus and/or Sapovirus capsid codingsequences are conveniently prepared using recombinant techniques. Asdiscussed below, VP1 polypeptide-encoding expression vectors of thepresent invention can include other polypeptide coding sequences ofinterest, for example, ORF1-encoded nonstructural proteins (e.g.,Norovirus Nterm, NTPase, p20, p22, VPg, Pro, and Pol; and Sapovirus p11,p28, NTPase, p32, VPg, Pro, and Pol) and minor structural proteins, suchas Norovirus VP2 and Sapovirus VP10. Such expression vectors can produceVLPs comprising VP1, as well as, any additional polypeptide of interest.

In certain embodiments, expression vectors may encode one or morestructural proteins from one or more genotypes and/or isolates ofNorovirus and Sapovirus. For example, expression vectors capable ofproducing VLPs can comprise one or more VP1 capsid proteins from one ormore isolates and/or genotypes of Norovirus and Sapovirus. In addition,expression vectors may further comprise coding sequences for one or moreminor structural proteins (e.g., VP2, VP10) from one or more isolatesand/or genotypes of Norovirus and Sapovirus.

Once coding sequences for the desired particle-forming polypeptides havebeen isolated or synthesized, they can be cloned into any suitablevector or replicon for expression. Numerous cloning vectors are known tothose of skill in the art, and the selection of an appropriate cloningvector is a matter of choice. See, generally, Ausubel et al, supra orSambrook et al, supra. The vector is then used to transform anappropriate host cell. Suitable recombinant expression systems include,but are not limited to, bacterial, baculovirus/insect, vaccinia, SemlikiForest virus (SFV), Alphaviruses (such as, Sindbis, Venezuelan EquineEncephalitis (VEE)), mammalian, yeast, plant, and Xenopus expressionsystems, well known in the art. Particularly preferred expressionsystems are mammalian cell lines, vaccinia, Sindbis, insect and yeastsystems.

For example, a number of mammalian cell lines are known in the art andinclude immortalized cell lines available from the American Type CultureCollection (A.T.C.C.), such as, but not limited to, Chinese hamsterovary (CHO) cells, 293 cells, HeLa cells, baby hamster kidney (BHK)cells, mouse myeloma (SB20), monkey kidney cells (COS), as well asothers. Similarly, bacterial hosts such as E. coli, Bacillus subtilis,and Streptococcus spp., will find use with the present expressionconstructs. Yeast hosts useful in the present invention include interalia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa,Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis,Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe andYarrowia lipolytica. See, e.g., Shuster et al. U.S. Pat. No. 6,183,985,herein incorporated by reference in its entirety. See also Example 1,which describes the expression of Norwalk virus VP1 and VP2 structuralproteins and production of viral particles in Saccharomyces cerevisiae.Insect cells for use with baculovirus expression vectors include, interalia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophilamelanogaster, Spodoptera frugiperda, and Trichoplusia ni. See, e.g.,Summers and Smith, Texas Agricultural Experiment Station Bulletin No.1555 (1987). See also Example 2, which describes the expression ofNorwalk virus VP1 and VP2 structural proteins and production of viralparticles in SF9 cells. Fungal hosts include, for example, Aspergillus.Plant hosts include tobacco, soybean, potato leaf and tuber tissues, andtomato fruit. See, e.g., Huang et al. (2005) Vaccine 23:1851-1858.

Viral vectors can be used for the production of particles in eucaryoticcells, such as those derived from the pox family of viruses, includingvaccinia virus and avian poxvirus. Additionally, a vaccinia basedinfection/transfection system, as described in Tomei et al., J. Virol.(1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993)74:1103-1113, will also find use with the present invention. In thissystem, cells are first infected in vitro with a vaccinia virusrecombinant that encodes the bacteriophage T7 RNA polymerase. Thispolymerase displays exquisite specificity in that it only transcribestemplates bearing T7 promoters. Following infection, cells aretransfected with the DNA of interest, driven by a T7 promoter. Thepolymerase expressed in the cytoplasm from the vaccinia virusrecombinant transcribes the transfected DNA into RNA which is thentranslated into protein by the host translational machinery.Alternately, T7 can be added as a purified protein or enzyme as in the“Progenitor” system (Studier and Moffatt, J. Mol. Biol. (1986)189:113-130). The method provides for high level, transient, cytoplasmicproduction of large quantities of RNA and its translation product(s).

Depending on the expression system and host selected, the VLPs areproduced by growing host cells transformed by an expression vector underconditions whereby the particle-forming polypeptide is expressed andVLPs can be formed. The selection of the appropriate growth conditionsis within the skill of the art.

If the VLPs are formed intracellularly, the cells are then disrupted,using chemical, physical or mechanical means, which lyse the cells yetkeep the VLPs substantially intact. Such methods are known to those ofskill in the art and are described in, e.g., Protein PurificationApplications: A Practical Approach, (E. L. V. Harris and S. Angal, Eds.,1990).

The particles are then isolated (or substantially purified) usingmethods that preserve the integrity thereof, such as, by densitygradient centrifugation, e.g., sucrose gradients, PEG-precipitation,pelleting, and the like (see, e.g., Kirnbauer et al. J. Virol. (1993)67:6929-6936), as well as standard purification techniques including,e.g., ion exchange and gel filtration chromatography.

In a further aspect, the present invention provides vectors and hostscells for production of mosaic VLPs comprising antigens from more thanone viral strain. Mosaic VLPs comprising capsid proteins from at leasttwo types of viruses, are produced by coexpressing capsid proteins fromat least two different genotypes and/or isolates of Norovirus and/orSapovirus in the same host cell. Coding sequences for capsidpolypeptides derived from at least two different genotypes and/orisolates of Norovirus and/or Sapovirus can be cloned into one or moreexpression vectors and coexpressed in cis or trans. In addition,expression vectors may further comprise coding sequences for one or moreminor structural proteins or nonstructural proteins from one or moreisolates and/or genotypes of Norovirus and/or Sapovirus.

Mosaic VLPs may comprise one or more VP1 polypeptides from multiplestrains of Norovirus (e.g., NV, SMV, and HV) or one or more VP1polypeptides from multiple strains of Sapovirus (e.g., Sapporo,London/29845, Parkville, Houston/90). Alternatively, mosaic VLPs maycomprise a combination of Norovirus and Sapovirus capsid proteins, suchmosaic VLPs comprising one or more VP1 polypeptides from one or morestrains of Norovirus and one or more VP1 polypeptides from one or morestrains of Sapovirus.

Mosaic VLPs can be produced by coexpression of multiple capsid proteinsusing any suitable recombinant expression system, such as thosedescribed above for expression of capsid proteins and production ofVLPs. In a preferred embodiment, capsid polypeptides can be expressed inan S. cerevisiae diploid strain produced by mating two haploid strains,each expressing different capsid proteins. See, e.g., InternationalPatent Publication WO 00/09699, herein incorporated by reference in itsentirety, which describes the production of mosaic VLPs in yeast byexpression of multiple capsid polypeptides using the episomal expressionvector pBS24.1 comprising an ADH2/GAPD glucose-repressible hybridpromoter.

VLPs of the present invention, including those comprising capsidproteins from a single viral strain and mosaic VLPs, can be used toelicit an immune response when administered to a subject. As discussedabove, the VLPs can comprise a variety of antigens in addition to theVP1 polypeptides (e.g., minor structural proteins and nonstructuralproteins). Purified VLPs, produced using the expression cassettes of thepresent invention, can be administered to a vertebrate subject, usuallyin the form of immunogenic compositions, such as vaccine compositions.Combination vaccines may also be used, where such immunogeniccompositions contain, for example, other proteins derived fromNoroviruses, Sapoviruses, or other organisms or nucleic acids encodingsuch antigens. Administration can take place using the VLPs formulatedalone or formulated with other antigens. Further, the VLPs can beadministered prior to, concurrent with, or subsequent to, delivery ofexpression cassettes for nucleic acid immunization (see below) and/ordelivery of other vaccines. Also, the site of VLP administration may bethe same or different as other immunogenic compositions that are beingadministered. Gene delivery can be accomplished by a number of methodsincluding, but are not limited to, immunization with DNA, alphavirusvectors, pox virus vectors, and vaccinia virus vectors.

F. Immunogenic Compositions

The invention also provides compositions comprising one or more of theimmunogenic nucleic acids, polypeptides, polyproteins multiepitopefusion proteins, and/or VLPs, described herein. Different polypeptides,polyproteins, and multiple epitope fusion proteins may be mixed togetherin a single formulation. Within such combinations, an antigen of theimmunogenic composition may be present in more than one polypeptide, ormultiple epitope polypeptide, or polyprotein.

The immunogenic compositions may comprise a mixture of polypeptides andnucleic acids, which in turn may be delivered using the same ordifferent vehicles. Antigens may be administered individually or incombination, in e.g., prophylactic (i.e., to prevent infection) ortherapeutic (to treat infection) immunogenic compositions. Theimmunogenic composition may be given more than once (e.g., a “prime”administration followed by one or more “boosts”) to achieve the desiredeffects. The same composition can be administered in one or more primingand one or more boosting steps. Alternatively, different compositionscan be used for priming and boosting.

The immunogenic compositions will generally include one or more“pharmaceutically acceptable excipients or vehicles” such as water,saline, glycerol, ethanol, etc. Additionally, auxiliary substances, suchas wetting or emulsifying agents, pH buffering substances, and the like,may be present in such vehicles.

Immunogenic compositions will typically, in addition to the componentsmentioned above, comprise one or more “pharmaceutically acceptablecarriers.” These include any carrier which does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Suitable carriers typically are large, slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, andlipid aggregates (such as oil droplets or liposomes). Such carriers arewell known to those of ordinary skill in the art. A composition may alsocontain a diluent, such as water, saline, glycerol, etc. Additionally,an auxiliary substance, such as a wetting or emulsifying agent, pHbuffering substance, and the like, may be present. A thorough discussionof pharmaceutically acceptable components is available in Gennaro (2000)Remington: The Science and Practice of Pharmacy. 20th ed., ISBN:0683306472.

Pharmaceutically acceptable salts can also be used in compositions ofthe invention, for example, mineral salts such as hydrochlorides,hydrobromides, phosphates, or sulfates, as well as salts of organicacids such as acetates, proprionates, malonates, or benzoates.Especially useful protein substrates are serum albumins, keyhole limpethemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanustoxoid, and other proteins well known to those of skill in the art.Compositions of the invention can also contain liquids or excipients,such as water, saline, glycerol, dextrose, ethanol, or the like, singlyor in combination, as well as substances such as wetting agents,emulsifying agents, or pH buffering agents. Antigens can also beadsorbed to, entrapped within or otherwise associated with liposomes andparticulate carriers such as PLG.

Antigens can be conjugated to a carrier protein in order to enhanceimmunogenicity. This is particularly useful in compositions in which asaccharide or carbohydrate antigen is used. See Ramsay et al. (2001)Lancet 357(9251):195-196; Lindberg (1999) Vaccine 17 Suppl 2:S28-36;Buttery & Moxon (2000) J R Coll Physicians Lond 34:163-168; Ahmad &Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii; Goldblatt(1998) J. Med. Microbiol. 47:563-567; European patent 0 477 508; U.S.Pat. No. 5,306,492; WO98/42721; Conjugate Vaccines (eds. Cruse et al.)ISBN 3805549326, particularly vol. 10:48-114; Hermanson (1996)Bioconjugate Techniques ISBN: 0123423368 or 012342335X.

Preferred carrier proteins are bacterial toxins or toxoids, such asdiphtheria or tetanus toxoids. The CRM₁₉₇ diphtheria toxoid isparticularly preferred. Other carrier polypeptides include the N.meningitidis outer membrane protein (EP-A-0372501), synthetic peptides(EP-A-0378881 and EP-A-0427347), heat shock proteins (WO 93/17712 and WO94/03208), pertussis proteins (WO 98/58668 and EP-A-0471177), protein Dfrom H. influenzae (WO 00/56360), cytokines (WO 91/01146), lymphokines,hormones, growth factors, toxin A or B from C. difficile (WO 00/61761),iron-uptake proteins, such as transferring (WO 01/72337), etc. Where amixture comprises capsular saccharide from both serigraphs A and C, itmay be preferred that the ratio (w/w) of MenA saccharide:MenC saccharideis greater than 1 (e.g., 2:1, 3:1, 4:1, 5:1, 10:1 or higher). Differentsaccharides can be conjugated to the same or different type of carrierprotein. Any suitable conjugation reaction can be used, with anysuitable linker where necessary.

Immunogenic compositions, preferably vaccines of the present inventionmay be administered in conjunction with other immunoregulatory agents.For example, a vaccine of the invention can include an adjuvant.Preferred adjuvants include, but are not limited to, one or more of thefollowing types of adjuvants described below.

A. Mineral Containing Compositions

Mineral containing compositions suitable for use as adjuvants in theinvention include mineral salts, such as aluminum salts and calciumsalts. The invention includes mineral salts such as hydroxides (e.g.oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates),sulfates, etc. (e.g. see chapters 8 & 9 of Vaccine Design . . . (1995)eds. Powell & Newman. ISBN: 030644867X. Plenum.), or mixtures ofdifferent mineral compounds (e.g. a mixture of a phosphate and ahydroxide adjuvant, optionally with an excess of the phosphate), withthe compounds taking any suitable form (e.g. gel, crystalline,amorphous, etc.), and with adsorption to the salt(s) being preferred.The mineral containing compositions may also be formulated as a particleof metal salt (WO00/23105).

Aluminum salts may be included in vaccines of the invention such thatthe dose of Al³⁺ is between 0.2 and 1.0 mg per dose.

In one embodiment the aluminum based adjuvant for use in the presentinvention is alum (aluminum potassium sulfate (AlK(SO₄)₂)), or an alumderivative, such as that formed in-situ by mixing an antigen inphosphate buffer with alum, followed by titration and precipitation witha base such as ammonium hydroxide or sodium hydroxide.

Another aluminum-based adjuvant for use in vaccine formulations of thepresent invention is aluminum hydroxide adjuvant (Al(OH)₃) orcrystalline aluminum oxyhydroxide (AlOOH), which is an excellentadsorbent, having a surface area of approximately 500 m²/g.Alternatively, aluminum phosphate adjuvant (AlPO₄) or aluminumhydroxyphosphate, which contains phosphate groups in place of some orall of the hydroxyl groups of aluminum hydroxide adjuvant is provided.Preferred aluminum phosphate adjuvants provided herein are amorphous andsoluble in acidic, basic and neutral media.

In another embodiment the adjuvant of the invention comprises bothaluminum phosphate and aluminum hydroxide. In a more particularembodiment thereof, the adjuvant has a greater amount of aluminumphosphate than aluminum hydroxide, such as a ratio of 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1 or greater than 9:1, by weight aluminumphosphate to aluminum hydroxide. More particular still, aluminum saltsin the vaccine are present at 0.4 to 1.0 mg per vaccine dose, or 0.4 to0.8 mg per vaccine dose, or 0.5 to 0.7 mg per vaccine dose, or about 0.6mg per vaccine dose.

Generally, the preferred aluminum-based adjuvant(s), or ratio ofmultiple aluminum-based adjuvants, such as aluminum phosphate toaluminum hydroxide is selected by optimization of electrostaticattraction between molecules such that the antigen carries an oppositecharge as the adjuvant at the desired pH. For example, aluminumphosphate adjuvant (iep=4) adsorbs lysozyme, but not albumin at pH 7.4.Should albumin be the target, aluminum hydroxide adjuvant would beselected (iep 11.4). Alternatively, pretreatment of aluminum hydroxidewith phosphate lowers its isoelectric point, making it a preferredadjuvant for more basic antigens.

B. Oil-Emulsions

Oil-emulsion compositions suitable for use as adjuvants in the inventioninclude squalene-water emulsions, such as MF59 (5% Squalene, 0.5% Tween80, and 0.5% Span 85, formulated into submicron particles using amicrofluidizer). See WO90/14837. See also, Podda, “The adjuvantedinfluenza vaccines with novel adjuvants: experience with theMF59-adjuvanted vaccine”, Vaccine (2001) 19: 2673-2680; Frey et al.,“Comparison of the safety, tolerability, and immunogenicity of aMF59-adjuvanted influenza vaccine and a non-adjuvanted influenza vaccinein non-elderly adults”, Vaccine (2003) 21:4234-4237. MF59 is used as theadjuvant in the FLUAD™ influenza virus trivalent subunit vaccine.

Particularly preferred adjuvants for use in the compositions aresubmicron oil-in-water emulsions. Preferred submicron oil-in-wateremulsions for use herein are squalene/water emulsions optionallycontaining varying amounts of MTP-PE, such as a submicron oil-in-wateremulsion containing 4-5% w/v squalene, 0.25-1.0% w/v Tween 80™(polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% Span85™(sorbitan trioleate), and, optionally,N-acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-huydroxyphosphosphoryloxy)-ethylamine(MTP-PE), for example, the submicron oil-in-water emulsion known as“MF59” (International Publication No. WO90/14837; U.S. Pat. Nos.6,299,884 and 6,451,325, and Ott et al., “MF59—Design and Evaluation ofa Safe and Potent Adjuvant for Human Vaccines” in Vaccine Design TheSubunit and Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.)Plenum Press, New York, 1995, pp. 277-296). MF59 contains 4-5% w/vSqualene (e.g. 4.3%), 0.25-0.5% w/v Tween 80™, and 0.5% w/v Span 85™ andoptionally contains various amounts of MTP-PE, formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton, Mass.). For example, MTP-PE may be present in anamount of about 0-500 μg/dose, more preferably 0-250 μg/dose and mostpreferably, 0-100 μg/dose. As used herein, the term “MF59-0” refers tothe above submicron oil-in-water emulsion lacking MTP-PE, while the termMF59-MTP denotes a formulation that contains MTP-PE. For instance,“MF59-100” contains 100 μg MTP-PE per dose, and so on. MF69, anothersubmicron oil-in-water emulsion for use herein, contains 4.3% w/vsqualene, 0.25% w/v Tween 80™, and 0.75% w/v Span 85™ and optionallyMTP-PE. Yet another submicron oil-in-water emulsion is MF75, also knownas SAF, containing 10% squalene, 0.4% Tween 80™, 5% pluronic-blockedpolymer L121, and thr-MDP, also microfluidized into a submicronemulsion. MF75-MTP denotes an MF75 formulation that includes MTP, suchas from 100-400 μg MTP-PE per dose.

Submicron oil-in-water emulsions, methods of'making the same andimmunostimulating agents, such as muramyl peptides, for use in thecompositions, are described in detail in International Publication No.WO90/14837 and U.S. Pat. Nos. 6,299,884 and 6,451,325.

Complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA)may also be used as adjuvants in the invention.

C. Saponin Formulations

Saponin formulations, may also be used as adjuvants in the invention.Saponins are a heterologous group of sterol glycosides and triterpenoidglycosides that are found in the bark, leaves, stems, roots and evenflowers of a wide range of plant species. Saponins isolated from thebark of the Quillaia saponaria Molina tree have been widely studied asadjuvants. Saponins can also be commercially obtained from Smilax ornata(sarsaprilla), Gypsophilla paniculata (brides veil), and Saponariaofficianalis (soap root). Saponin adjuvant formulations include purifiedformulations, such as QS21, as well as lipid formulations, such asISCOMs.

Saponin compositions have been purified using High Performance ThinLayer Chromatography (HP-TLC) and Reversed Phase High Performance LiquidChromatography (RP-HPLC), Specific purified fractions using thesetechniques have been identified, including QS7, QS17, QS18, QS21, QH-A,QH-B and QH-C. Preferably, the saponin is QS21. A method of productionof QS21 is disclosed in U.S. Pat. No. 5,057,540. Saponin formulationsmay also comprise a sterol, such as cholesterol (see WO96/33739).

Combinations of saponins and cholesterols can be used to form uniqueparticles called Immunostimulating Complexes (ISCOMs). ISCOMs typicallyalso include a phospholipid such as phosphatidylethanolamine orphosphatidylcholine. Any known saponin can be used in ISCOMs.Preferably, the ISCOM includes one or more of Quil A, QHA and QHC.ISCOMs are further described in EP0109942, WO96/11711 and WO96/33739.Optionally, the ISCOMS may be devoid of (an) additional detergent(s).See WO00/07621.

A review of the development of saponin based adjuvants can be found inBarr, et al., “ISCOMs and other saponin based adjuvants”, Advanced DrugDelivery Reviews (1998) 32:247-271. See also Sjolander, et al., “Uptakeand adjuvant activity of orally delivered saponin and ISCOM vaccines”,Advanced Drug Delivery Reviews (1998) 32:321-338.

D. Virosomes and Virus Like Particles (VLPs)

Virosomes and Virus Like Particles (VLPs) can also be used as adjuvantsin the invention. These structures generally contain one or moreproteins from a virus optionally combined or formulated with aphospholipid. They are generally non-pathogenic, non-replicating andgenerally do not contain any of the native viral genome. The viralproteins may be recombinantly produced or isolated from whole viruses.These viral proteins suitable for use in virosomes or VLPs includeproteins derived from influenza virus (such as HA or NA), Hepatitis Bvirus (such as core or capsid proteins), Hepatitis E virus, measlesvirus, Sindbis virus, Rotavirus, Foot-and-Mouth Disease virus,Retrovirus, Norwalk virus, human Papilloma virus, HIV, RNA-phages,Qβ-phage (such as coat proteins), GA-phage, fr-phage; AP205 phage, andTy (such as retrotransposon Ty protein p1). VLPs are discussed furtherin WO03/024480, WO03/024481, and Niikura et al., “Chimeric RecombinantHepatitis E Virus-Like Particles as an Oral Vaccine Vehicle PresentingForeign Epitopes”, Virology (2002) 293:273-280; Lenz et al.,“Papillomarivurs-Like Particles Induce Acute Activation of DendriticCells”, Journal of Immunology (2001) 5246-5355; Pinto, et al., “CellularImmune Responses to Human Papillomavirus (HPV)-16 μl Healthy VolunteersImmunized with Recombinant HPV-16 L1 Virus-Like Particles”, Journal ofInfectious Diseases (2003) 188:327-338; and Gerber et al., “HumanPapillomavrisu Virus-Like Particles Are Efficient Oral Immunogens whenCoadministered with Escherichia coli Heat-Labile Entertoxin Mutant R192Gor CpG”, Journal of Virology (2001) 75(10):4752-4760. Virosomes arediscussed further in, for example, Gluck et al., “New TechnologyPlatforms in the Development of Vaccines for the Future”, Vaccine (2002)20:B10-B16. Immunopotentiating reconstituted influenza virosomes (IRIV)are used as the subunit antigen delivery system in the intranasaltrivalent INFLEXAL™ product {Mischler & Metcalfe (2002) Vaccine 20 Suppl5:B17-23} and the INFLUVAC PLUS™ product.

E. Bacterial or Microbial Derivatives

Adjuvants suitable for use in the invention include bacterial ormicrobial derivatives such as:

(1) Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide (LPS)

Such derivatives include Monophosphoryl lipid A (MPL) and 3-O-deacylatedMPL (3dMPL). 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipidA with 4, 5 or 6 acylated chains. A preferred “small particle” form of 3De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689 454. Such“small particles” of 3dMPL are small enough to be sterile filteredthrough a 0.22 micron membrane (see EP 0 689 454). Other non-toxic LPSderivatives include monophosphoryl lipid A mimics, such as aminoalkylglucosaminide phosphate derivatives e.g. RC-529. See Johnson et al.(1999) Bioorg Med Chem Lett 9:2273-2278.

(2) Lipid A Derivatives

Lipid A derivatives include derivatives of lipid A from Escherichia colisuch as OM-174. OM-174 is described for example in Meraldi et al.,“OM-174, a New Adjuvant with a Potential for Human Use, Induces aProtective Response with Administered with the Synthetic C-TerminalFragment 242-310 from the circumsporozoite protein of Plasmodiumberghei”, Vaccine (2003) 21:2485-2491; and Pajak, et al., “The AdjuvantOM-174 induces both the migration and maturation of murine dendriticcells in vivo”, Vaccine (2003) 21:836-842.

(3) Immunostimulatory Oligonucleotides

Immunostimulatory oligonucleotides suitable for use as adjuvants in theinvention include nucleotide sequences containing a CpG motif (asequence containing an unmethylated cytosine followed by guanosine andlinked by a phosphate bond). Bacterial double stranded RNA oroligonucleotides containing palindromic or poly(dG) sequences have alsobeen shown to be immunostimulatory.

The CpG's can include nucleotide modifications/analogs such asphosphorothioate modifications and can be double-stranded orsingle-stranded. Optionally, the guanosine may be replaced with ananalog such as 2′-deoxy-7-deazaguanosine. See Kandimalla, et al.,“Divergent synthetic nucleotide motif recognition pattern: design anddevelopment of potent immunomodulatory oligodeoxyribonucleotide agentswith distinct cytokine induction profiles”, Nucleic Acids Research(2003) 31(9): 2393-2400; WO02/26757 and WO99/62923 for examples ofpossible analog substitutions. The adjuvant effect of CpGoligonucleotides is further discussed in Krieg, “CpG motifs: the activeingredient in bacterial extracts?”, Nature Medicine (2003) 9(7):831-835; McCluskie, et al., “Parenteral and mucosal prime-boostimmunization strategies in mice with hepatitis B surface antigen and CpGDNA”, FEMS Immunology and Medical Microbiology (2002) 32:179-185;WO98/40100; U.S. Pat. No. 6,207,646; U.S. Pat. No. 6,239,116 and U.S.Pat. No. 6,429,199.

The CpG sequence may be directed to TLR9, such as the motif GTCGTT orTTCGTT. See Kandimalla, et al., “Toll-like receptor 9: modulation ofrecognition and cytokine induction by novel synthetic CpG DNAs”,Biochemical Society Transactions (2003) 31 (part 3): 654-658. The CpGsequence may be specific for inducing a Th1 immune response, such as aCpG-A ODN, or it may be more specific for inducing a B cell response,such a CpG-B ODN. CpG-A and CpG-B ODNs are discussed in Blackwell, etal., “CpG-A-Induced Monocyte IFN-gamma-Inducible Protein-10 Productionis Regulated by Plasmacytoid Dendritic Cell Derived IFN-alpha”, J.Immunol. (2003) 170(8):4061-4068; Krieg, “From A to Z on CpG”, TRENDS inImmunology (2002) 23(2): 64-65 and WO01/95935. Preferably, the CpG is aCpG-A ODN.

Preferably, the CpG oligonucleotide is constructed so that the 5′ end isaccessible for receptor recognition. Optionally, two CpG oligonucleotidesequences may be attached at their 3′ ends to form “immunomers”. See,for example, Kandimalla, et al., “Secondary structures in CpGoligonucleotides affect immunostimulatory activity”, BBRC (2003)306:948-953; Kandimalla, et al., “Toll-like receptor 9: modulation ofrecognition and cytokine induction by novel synthetic GpG DNAs”,Biochemical Society Transactions (2003) 31(part 3):664-658; Bhagat etal., “CpG penta- and hexadeoxyribonucleotides as potent immunomodulatoryagents” BBRC (2003) 300:853-861 and WO03/035836.

(4) ADP-ribosylating Toxins and Detoxified Derivatives Thereof.

Bacterial ADP-ribosylating toxins and detoxified derivatives thereof maybe used as adjuvants in the invention. Preferably, the protein isderived from E. coli (i.e., E. coli heat labile enterotoxin “LT),cholera (“CT”), or pertussis (“PT”). The use of detoxifiedADP-ribosylating toxins as mucosal adjuvants is described in WO95/17211and as parenteral adjuvants in WO98/42375. Preferably, the adjuvant is adetoxified LT mutant such as LT-K63, LT-R72, and LTR192G. The use ofADP-ribosylating toxins and detoxified derivatives thereof, particularlyLT-K63 and LT-R72, as adjuvants can be found in the followingreferences: Beignon, et al., “The LTR72Mutant of Heat-Labile Enterotoxinof Escherichia coli Enhances the Ability of Peptide Antigens to ElicitCD4+ T Cells and Secrete Gamma Interferon after Coapplication onto BareSkin”, Infection and Immunity (2002) 70(6):3012-3019; Pizza, et al.,“Mucosal vaccines: non toxic derivatives of LT and CT as mucosaladjuvants”, Vaccine (2001) 19:2534-2541; Pizza, et al., “LTK63 andLTR72, two mucosal adjuvants ready for clinical trials” Int. J. Med.Microbiol. (2000) 290(4-5):455-461; Scharton-Kersten et al.,“Transcutaneous Immunization with Bacterial ADP-Ribosylating Exotoxins,Subunits and Unrelated Adjuvants”, Infection and Immunity (2000)68(9):5306-5313; Ryan et al., “Mutants of Escherichia coli Heat-LabileToxin Act as Effective Mucosal Adjuvants for Nasal Delivery of anAcellular Pertussis Vaccine: Differential Effects of the Nontoxic ABComplex and Enzyme Activity on Th1 and Th2 Cells” Infection and Immunity(1999) 67(12):6270-6280; Partidos et al., “Heat-labile enterotoxin ofEscherichia coli and its site-directed mutant LT-K63 enhance theproliferative and cytotoxic T-cell responses to intranasallyco-immunized synthetic peptides”, Immunol. Lett. (1999) 67(3):209-216;Peppoloni et al., “Mutants of the Escherichia coli heat-labileenterotoxin as safe and strong adjuvants for intranasal delivery ofvaccines”, Vaccines (2003) 2(2):285-293; and Pine et al., (2002)“Intranasal immunization with influenza vaccine and a detoxified mutantof heat labile enterotoxin from Escherichia coli (LTK63)” J. ControlRelease (2002) 85(1-3):263-270. Numerical reference for amino acidsubstitutions is preferably based on the alignments of the A and Bsubunits of ADP-ribosylating toxins set forth in Domenighini et al.,Mol. Microbiol. (1995) 15(6):1165-1167.

F. Bioadhesives and Mucoadhesives

Bioadhesives and mucoadhesives may also be used as adjuvants in theinvention. Suitable bioadhesives include esterified hyaluronic acidmicrospheres (Singh et al. (2001) J. Cont. Rele. 70:267-276) ormucoadhesives such as cross-linked derivatives of polyacrylic acid,polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides andcarboxymethylcellulose. Chitosan and derivatives thereof may also beused as adjuvants in the invention. E.g. WO99/27960.

G. Microparticles

Microparticles may also be used as adjuvants in the invention.Microparticles (i.e. a particle of ˜100 nm to ˜150 μm in diameter, morepreferably ˜200 nm to ˜30 μm in diameter, and most preferably ˜500 nm to˜10 μm in diameter) formed from materials that are biodegradable andnon-toxic (e.g. a poly(α-hydroxy acid), a polyhydroxybutyric acid, apolyorthoester, a polyanhydride, a polycaprolactone, etc.), withpoly(lactide-co-glycolide) are preferred, optionally treated to have anegatively-charged surface (e.g. with SDS) or a positively-chargedsurface (e.g. with a cationic detergent, such as CTAB).

H. Liposomes

Examples of liposome formulations suitable for use as adjuvants aredescribed in U.S. Pat. No. 6,090,406, U.S. Pat. No. 5,916,588, and EP 0626 169.

I. Polyoxyethylene ether and Polyoxyethylene Ester Formulations

Adjuvants suitable for use in the invention include polyoxyethyleneethers and polyoxyethylene esters. WO99/52549. Such formulations furtherinclude polyoxyethylene sorbitan ester surfactants in combination withan octoxynol (WO01/21207) as well as polyoxyethylene alkyl ethers orester surfactants in combination with at least one additional non-ionicsurfactant such as an octoxynol (WO01/21152).

Preferred polyoxyethylene ethers are selected from the following group:polyoxyethylene-9-lauryl ether (laureth 9), polyoxyethylene-9-steorylether, polyoxytheylene-8-steoryl ether, polyoxyethylene-4-lauryl ether,polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether.

J. Polyphosphazene (PCPP)

PCPP formulations are described, for example, in Andrianov et al.,“Preparation of hydrogel microspheres by coacervation of aqueouspolyphophazene solutions”, Biomaterials (1998) 19(1-3):109-115 and Payneet al., “Protein Release from Polyphosphazene Matrices”, Adv. Drug.Delivery Review (1998) 31(3):185-196.

K. Muramyl Peptides

Examples of muramyl peptides suitable for use as adjuvants in theinvention include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-1-alanyl-d-isoglutamine (nor-MDP), andN-acetylmuramyl-1-alanyl-d-isoglutaminyl-1-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE).

L. Imidazoquinoline Compounds

Examples of imidazoquinoline compounds suitable for use adjuvants in theinvention include Imiquimod and its analogues, described further inStanley, “Imiquimod and the imidazoquinolines: mechanism of action andtherapeutic potential” Clin Exp Dermatol (2002) 27(7):571-577; Jones,“Resiquimod 3M”, Curr Opin Investig Drugs (2003) 4(2):214-218; and U.S.Pat. Nos. 4,689,338, 5,389,640, 5,268,376, 4,929,624, 5,266,575,5,352,784, 5,494,916, 5,482,936, 5,346,905, 5,395,937, 5,238,944, and5,525,612.

M. Thiosemicarbazone Compounds.

Examples of thiosemicarbazone compounds, as well as methods offormulating, manufacturing, and screening for compounds all suitable foruse as adjuvants in the invention include those described in WO04/60308.The thiosemicarbazones are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

N. Tryptanthrin Compounds.

Examples of tryptanthrin compounds, as well as methods of formulating,manufacturing, and screening for compounds all suitable for use asadjuvants in the invention include those described in WO04/64759. Thetryptanthrin compounds are particularly effective in the stimulation ofhuman peripheral blood mononuclear cells for the production ofcytokines, such as TNF-α.

The invention may also comprise combinations of aspects of one or moreof the adjuvants identified above. For example, the following adjuvantcompositions may be used in the invention:

(1) a saponin and an oil-in-water emulsion (WO99/11241);

(2) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g. 3dMPL) (seeWO94/00153);

(3) a saponin (e.g., QS21)+a non-toxic LPS derivative (e.g. 3dMPL)+acholesterol;

(4) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+a sterol)(WO98/57659);

(5) combinations of 3dMPL with, for example, QS21 and/or oil-in-wateremulsions (See European patent applications 0835318, 0735898 and0761231);

(6) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blockpolymer L121, and thr-MDP, either microfluidized into a submicronemulsion or vortexed to generate a larger particle size emulsion.

(7) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2%Squalene, 0.2% Tween 80, and one or more bacterial cell wall componentsfrom the group consisting of monophosphorylipid A (MPL), trehalosedimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS(Detox™); and(8) one or more mineral salts (such as an aluminum salt)+a non-toxicderivative of LPS (such as 3dPML).(9) one or more mineral salts (such as an aluminum salt) and one or moreimmunostimulatory oligonucleotides (such as a nucleotide sequenceincluding a CpG motif) and one or more detoxified ADP-ribosylatingtoxins (such as LT-K63 and LT-R72).O. Human Immunomodulators

Human immunomodulators suitable for use as adjuvants in the inventioninclude cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-6,IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colonystimulating factor, and tumor necrosis factor.

Aluminum salts and MF59 are preferred adjuvants for use with injectableNorovirus and Sapovirus vaccines. Bacterial toxins and bioadhesives arepreferred adjuvants for use with mucosally-delivered vaccines, such asnasal vaccines.

The contents of all of the above cited patents, patent applications andjournal articles are incorporated by reference as if set forth fullyherein.

Additional Antigens

Compositions of the invention optionally can comprise one or moreadditional polypeptide antigens which are not derived from Norovirus orSapovirus proteins. Such antigens include bacterial, viral, or parasiticantigens.

In some embodiments, a Norovirus or Sapovirus antigen is combined withone or more antigens which are useful in a pediatric vaccine. Suchantigens are well known in the art and include, but are not limited to,antigens derived from a bacteria or virus, such as Orthomyxovirus(influenza), Pneumovirus (RSV), Paramyxovirus (PIV and Mumps),Morbillivirus (measles), Togavirus (Rubella), Enterovirus HBV,Coronavirus (SARS), and Varicella-zoster virus (VZV), Epstein Barr virus(EBV), Streptococcus pneumoniae, Neisseria meningitides, Streptococcuspyogenes (Group A Streptococcus), Moraxella catarrhalis, Bordetellapertussis, Staphylococcus aureus, Clostridium tetani (Tetanus),Cornynebacterium diphtheriae (Diphtheria), Haemophilus influenzae B(Hib), Pseudomonas aeruginosa, Streptococcus agalactiae (Group BStreptococcus), and E. coli.

In other embodiments, a Norovirus or Sapovirus antigen is combined withone or more antigens useful in a vaccine designed to protect elderly orimmunocompromised individuals. Antigens of this type are well known inthe art and include, but are not limited to, Neisseria meningitides,Streptococcus pneumoniae, Streptococcus pyogenes (Group AStreptococcus), Moraxella catarrhalis, Bordetella pertussis,Staphylococcus aureus, Staphylococcus epidermis, Clostridium tetani(Tetanus), Cornynebacterium diphtheriae (Diphtheria), Haemophilusinfluenzae B (Hib), Pseudomonas aeruginosa, Legionella pneumophila,Streptococcus agalactiae (Group B Streptococcus), Enterococcus faecalis,Helicobacter pylori, Clamydia pneumoniae, Orthomyxovirus (influenza),Pneumovirus (RSV), Paramyxovirus (PIV and Mumps), Morbillivirus(measles), Togavirus (Rubella), Enterovirus (polio), HBV, Coronavirus(SARS), Varicella-zoster virus (VZV), Epstein Barr virus (EBV),Cytomegalovirus (CMV).

In other embodiments, a Norovirus or Sapovirus antigen is combined withone or more antigens which are useful in a vaccine designed to protectindividuals against pathogens that cause diarrheal diseases. Suchantigens include, but are not limited to, rotavirus, Shigella spp.,enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, andCampylobacter jejuni antigens. In a preferred embodiment, one or moreNorovirus antigens derived from Norwalk virus, Snow Mountain virus,and/or Hawaii virus are combined with a rotavirus antigen in animmunogenic composition.

Antigens for use with the invention include, but are not limited to, oneor more of the following antigens set forth below, or antigens derivedfrom one or more of the pathogens set forth below:

A. BACTERIAL ANTIGENS

Bacterial antigens suitable for use in the invention include proteins,polysaccharides, lipopolysaccharides, and outer membrane vesicles whichmay be isolated, purified or derived from a bacteria. In addition,bacterial antigens may include bacterial lysates and inactivatedbacteria formulations. Bacteria antigens may be produced by recombinantexpression. Bacterial antigens preferably include epitopes which areexposed on the surface of the bacteria during at least one stage of itslife cycle. Bacterial antigens are preferably conserved across multipleserotypes. Bacterial antigens include antigens derived from one or moreof the bacteria set forth below as well as the specific antigensexamples identified below.

Neisseria meningitides: Meningitides antigens may include proteins (suchas those identified in References 1-7), saccharides (including apolysaccharide, oligosaccharide or lipopolysaccharide), orouter-membrane vesicles (References 8, 9, 10, 11) purified or derivedfrom N. meningitides serogroup such as A, C, W135, Y, and/or B.Meningitides protein antigens may be selected from adhesions,autotransporters, toxins, Fe acquisition proteins, and membraneassociated proteins (preferably integral outer membrane protein).

Streptococcus pneumoniae: Streptococcus pneumoniae antigens may includea saccharide (including a polysaccharide or an oligosaccharide) and/orprotein from Streptococcus pneumoniae. Saccharide antigens may beselected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F,14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and 33F. Protein antigens maybe selected from a protein identified in WO 98/18931, WO 98/18930, U.S.Pat. No. 6,699,703, U.S. Pat. No. 6,800,744, WO 97/43303, and WO97/37026. Streptococcus pneumoniae proteins may be selected from thePoly Histidine Triad family (PhtX), the Choline Binding Protein family(CbpX), CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytXtruncate chimeric proteins, pneumolysin (Ply), PspA, PsaA, Sp128, Sp101,Sp130, Sp125 or Sp133.

Streptococcus pyogenes (Group A Streptococcus): Group A Streptococcusantigens may include a protein identified in WO 02/34771 or WO2005/032582 (including GAS 40), fusions of fragments of GAS M proteins(including those described in WO 02/094851, and Dale, Vaccine (1999)17:193-200, and Dale, Vaccine 14(10): 944-948), fibronectin bindingprotein (Sfb1), Streptococcal heme-associated protein (Shp), andStreptolysin S (SagA).

Moraxella catarrhalis: Moraxella antigens include antigens identified inWO 02/18595 and WO 99/58562, outer membrane protein antigens (HMW-OMP),C-antigen, and/or LPS.

Bordetella pertussis: Pertussis antigens include petussis holotoxin (PT)and filamentous haemagglutinin (FHA) from B. pertussis, optionally alsocombination with pertactin and/or agglutinogens 2 and 3 antigen.

Staphylococcus aureus: Staph aureus antigens include S. aureus type 5and 8 capsular polysaccharides optionally conjugated to nontoxicrecombinant Pseudomonas aeruginosa exotoxin A, such as StaphVAX™, orantigens derived from surface proteins, invasins (leukocidin, kinases,hyaluronidase), surface factors that inhibit phagocytic engulfment(capsule, Protein A), carotenoids, catalase production, Protein A,coagulase, clotting factor, and/or membrane-damaging toxins (optionallydetoxified) that lyse eukaryotic cell membranes (hemolysins, leukotoxin,leukocidin).

Staphylococcus epidermis: S. epidermidis antigens includeslime-associated antigen (SAA).

Clostridium tetani (Tetanus): Tetanus antigens include tetanus toxoid(TT), preferably used as a carrier protein in conjunction/conjugatedwith the compositions of the present invention.

Cornynebacterium diphtheriae (Diphtheria): Diphtheria antigens includediphtheria toxin, preferably detoxified, such as CRM₁₉₇. Additionallyantigens capable of modulating, inhibiting or associated with ADPribosylation are contemplated forcombination/co-administration/conjugation with the compositions of thepresent invention. The diphtheria toxoids may be used as carrierproteins.

Haemophilus influenzae B (Hib): Hib antigens include a Hib saccharideantigen.

Pseudomonas aeruginosa: Pseudomonas antigens include endotoxin A, Wzzprotein, P. aeruginosa LPS, more particularly LPS isolated from PAO1 (O5serotype), and/or Outer Membrane Proteins, including Outer MembraneProteins F (OprF) (Infect Immun. 2001 May; 69(5): 3510-3515).

Legionella pneumophila. Bacterial antigens may be derived fromLegionella pneumophila.

Streptococcus agalactiae (Group B Streptococcus): Group B Streptococcusantigens include a protein or saccharide antigen identified in WO02/34771, WO 03/093306, WO 04/041157, or WO 2005/002619 (includingproteins GBS 80, GBS 104, GBS 276 and GBS 322, and including saccharideantigens derived from serotypes Ia, Ib, Ia/c, II, III, IV, V, VI, VIIand VIII).

Neiserria gonorrhoeae: Gonorrhoeae antigens include Por (or porin)protein, such as PorB (see Zhu et al., Vaccine (2004) 22:660-669), atransferring binding protein, such as TbpA and TbpB (See Price et al.,Infection and Immunity (2004) 71(1):277-283), a opacity protein (such asOpa), a reduction-modifiable protein (Rmp), and outer membrane vesicle(OMV) preparations (see Plante et al., J Infectious Disease (2000)182:848-855), also see e.g. WO99/24578, WO99/36544, WO99/57280,WO02/079243).

Chlamydia trachomatis: Chlamydia trachomatis antigens include antigensderived from serotypes A, B, Ba and C (agents of trachoma, a cause ofblindness), serotypes L₁, L₂ & L₃ (associated with Lymphogranulomavenereum), and serotypes, D-K. Chlamydia trachomas antigens may alsoinclude an antigen identified in WO 00/37494, WO 03/049762, WO03/068811, or WO 05/002619; including PepA (CT045), LcrE (CT089), ArtJ(CT381), DnaK (CT396), CT398, OmpH-like (CT242), L7/L12 (CT316), OmcA(CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), and MurG(CT761).

Treponema pallidum (Syphilis): Syphilis antigens include TmpA antigen.

Haemophilus ducreyi (causing chancroid): Ducreyi antigens include outermembrane protein (DsrA).

Enterococcus faecalis or Enterococcus faecium: Antigens include atrisaccharide repeat or other Enterococcus derived antigens provided inU.S. Pat. No. 6,756,361.

Helicobacter pylori: H pylori antigens include Cag, Vac, Nap, HopX, HopYand/or urease antigen.

Staphylococcus saprophyticus: Antigens include the 160 kDa hemagglutininof S. saprophyticus antigen.

Yersinia enterocolitica Antigens include LPS (Infect Immun. 2002 August;70(8): 4414).

E. coli: E. coli antigens may be derived from enterotoxigenic E. coli(ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli(DAEC), enteropathogehic E. coli (EPEC), and/or enterohemorrhagic E.coli (EHEC).

Bacillus anthracis (anthrax): B. anthracis antigens are optionallydetoxified and may be selected from A-components (lethal factor (LF) andedema factor (EF)), both of which can share a common B-component knownas protective antigen (PA).

Yersinia pestis (plague): Plague antigens include F1 capsular antigen(Infect Immun. 2003 January; 71(1)): 374-383, LPS (Infect Immun. 1999October; 67(10): 5395), Yersinia pestis V antigen (Infect Immun. 1997November; 65(11): 4476-4482).

Mycobacterium tuberculosis: Tuberculosis antigens include lipoproteins,LPS, BCG antigens, a fusion protein of antigen 85B (Ag85B) and/or ESAT-6optionally formulated in cationic lipid vesicles (Infect Immun. 2004October; 72(10): 6148), Mycobacterium tuberculosis (Mtb) isocitratedehydrogenase associated antigens (Proc Natl Acad Sci USA. 2004 Aug. 24;101(34): 12652), and/or MPT51 antigens (Infect Immun. 2004 July; 72(7):3829).

Rickettsia: Antigens include outer membrane proteins, including theouter membrane protein A and/or B (OmpB) (Biochim Biophys Acta. 2004Nov. 1; 1702(2):145), LPS, and surface protein antigen (SPA) (JAutoimmun. 1989 June; 2 Suppl:81).

Listeria monocytogenes. Bacterial antigens may be derived from Listeriamonocytogenes.

Chlamydia pneumoniae: Antigens include those identified in WO 02/02606.

Vibrio cholerae: Antigens include proteinase antigens, LPS, particularlylipopolysaccharides of Vibrio cholerae II, O1 Inaba O-specificpolysaccharides, V. cholera 0139, antigens of IEM108 vaccine (InfectImmun. 2003 October; 71(10):5498-504), and/or Zonula occludens toxin(Zot).

Salmonella typhi (typhoid fever): Antigens include capsularpolysaccharides preferably conjugates (Vi, i.e. vax-TyVi).

Borrelia burgdorferi (Lyme disease): Antigens include lipoproteins (suchas OspA, OspB, Osp C and Osp D), other surface proteins such asOspE-related proteins (Erps), decorin-binding proteins (such as DbpA),and antigenically variable VI proteins, such as antigens associated withP39 and P13 (an integral membrane protein, Infect Immun. 2001 May;69(5): 3323-3334), V1sE Antigenic Variation Protein (J Clin Microbiol.1999 December; 37(12): 3997).

Porphyromonas gingivalis: Antigens include P. gingivalis outer membraneprotein (OMP).

Klebsiella: Antigens include an OMP, including OMP A, or apolysaccharide optionally conjugated to tetanus toxoid.

Further bacterial antigens of the invention may be capsular antigens,polysaccharide antigens or protein antigens of any of the above. Furtherbacterial antigens may also include an outer membrane vesicle (OMV)preparation. Additionally, antigens include live, attenuated, and/orpurified versions of any of the aforementioned bacteria. The antigens ofthe present invention may be derived from gram-negative or gram-positivebacteria. The antigens of the present invention may be derived fromaerobic or anaerobic bacteria.

Additionally, any of the above bacterial-derived saccharides(polysaccharides, LPS, LOS or oligosaccharides) can be conjugated toanother agent or antigen, such as a carrier protein (for exampleCRM₁₉₇). Such conjugation may be direct conjugation effected byreductive amination of carbonyl moieties on the saccharide to aminogroups on the protein, as provided in U.S. Pat. No. 5,360,897 and Can JBiochem Cell Biol. 1984 May; 62(5):270-5. Alternatively, the saccharidescan be conjugated through a linker, such as, with succinamide or otherlinkages provided in Bioconjugate Techniques, 1996 and CRC, Chemistry ofProtein Conjugation and Cross-Linking, 1993.

B. VIRAL ANTIGENS

Viral antigens suitable for use in the invention include inactivated (orkilled) virus, attenuated virus, split virus formulations, purifiedsubunit formulations, viral proteins which may be isolated, purified orderived from a virus, and Virus Like Particles (VLPs). Viral antigensmay be derived from viruses propagated on cell culture or othersubstrate. Alternatively, viral antigens may be expressed recombinantly.Viral antigens preferably include epitopes which are exposed on thesurface of the virus during at least one stage of its life cycle. Viralantigens are preferably conserved across multiple serotypes or isolates.Viral antigens include antigens derived from one or more of the Virusesset forth below as well as the specific antigens examples identifiedbelow.

Orthomyxovirus: Viral antigens may be derived from an Orthomyxovirus,such as Influenza A, B and C. Orthomyxovirus antigens may be selectedfrom one or more of the viral proteins, including hemagglutinin (HA),neuraminidase (NA), nucleoprotein (NP), matrix protein (M1), membraneprotein (M2), one or more of the transcriptase components (PB1, PB2 andPA). Preferred antigens include HA and NA.

Influenza antigens may be derived from interpandemic (annual) flustrains. Alternatively influenza antigens may be derived from strainswith the potential to cause pandemic a pandemic outbreak (i.e.,influenza strains with new haemagglutinin compared to the haemagglutininin currently circulating strains, or influenza strains which arepathogenic in avian subjects and have the potential to be transmittedhorizontally in the human population, or influenza strains which arepathogenic to humans).

Paramyxoviridae viruses: Viral antigens may be derived fromParamyxoviridae viruses, such as Pneumoviruses (RSV), Paramyxoviruses(PIV) and Morbilliviruses (Measles).

Pneumovirus: Viral antigens may be derived from a Pneumovirus, such asRespiratory syncytial virus (RSV), Bovine respiratory syncytial virus,Pneumonia virus of mice, and Turkey rhinotracheitis virus. Preferably,the Pneumovirus is RSV. Pneumovirus antigens may be selected from one ormore of the following proteins, including surface proteins Fusion (F),Glycoprotein (G) and Small Hydrophobic protein (SH), matrix proteins Mand M2, nucleocapsid proteins N, P and L and nonstructural proteins NS1and NS2. Preferred Pneumovirus antigens include F, G and M. See e.g., JGen Virol. 2004 November; 85(Pt 11):3229). Pneumovirus antigens may alsobe formulated in or derived from chimeric viruses. For example, chimericRSV/PIV viruses may comprise components of both RSV and PIV.

Paramyxovirus: Viral antigens may be derived from a Paramyxovirus, suchas Parainfluenza virus types 1-4 (PIV), Mumps, Sendai viruses, Simianvirus 5, Bovine parainfluenza virus and Newcastle disease virus.Preferably, the Paramyxovirus is PIV or Mumps. Paramyxovirus antigensmay be selected from one or more of the following proteins:Hemagglutinin-Neuraminidase (HN), Fusion proteins F1 and F2,Nucleoprotein (NP), Phosphoprotein (P), Large protein (L), and Matrixprotein (M). Preferred Paramyxovirus proteins include HN, F1 and F2.Paramyxovirus antigens may also be formulated in or derived fromchimeric viruses. For example, chimeric RSV/PIV viruses may comprisecomponents of both RSV and PIV. Commercially available mumps vaccinesinclude live attenuated mumps virus, in either a monovalent form or incombination with measles and rubella vaccines (MMR).

Morbillivirus: Viral antigens may be derived from a Morbillivirus, suchas Measles. Morbillivirus antigens may be selected from one or more ofthe following proteins: hemagglutinin (H), Glycoprotein (G), Fusionfactor (F), Large protein (L), Nucleoprotein (NP), Polymerasephosphoprotein (P), and Matrix (M). Commercially available measlesvaccines include live attenuated measles virus, typically in combinationwith mumps and rubella (MMR).

Picornavirus: Viral antigens may be derived from Picornaviruses, such asEnteroviruses, Rhinoviruses, Heparnavirus, Cardioviruses andAphthoviruses. Antigens derived from Enteroviruses, such as Poliovirusare preferred.

Enterovirus: Viral antigens may be derived from an Enterovirus, such asPoliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24,Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus) types 1 to 9, 11to 27 and 29 to 34 and Enterovirus 68 to 71. Preferably, the Enterovirusis poliovirus. Enterovirus antigens are preferably selected from one ormore of the following Capsid proteins VP1, VP2, VP3 and VP4.Commercially available polio vaccines include Inactivated Polio Vaccine(IPV) and Oral poliovirus vaccine (OPV).

Heparnavirus: Viral antigens may be derived from an Heparnavirus, suchas Hepatitis A virus (HAV). Commercially available HAV vaccines includeinactivated HAV vaccine.

Togavirus: Viral antigens may be derived from a Togavirus, such as aRubivirus, an Alphavirus, or an Arterivirus. Antigens derived fromRubivirus, such as Rubella virus, are preferred. Togavirus antigens maybe selected from E1, E2, E3, C, NSP-1, NSPO-2, NSP-3 or NSP-4. Togavirusantigens are preferably selected from E1, E2 or E3. Commerciallyavailable Rubella vaccines include a live cold-adapted virus, typicallyin combination with mumps and measles vaccines (MMR).

Flavivirus: Viral antigens may be derived from a Flavivirus, such asTick-borne encephalitis (TBE), Dengue (types 1, 2, 3 or 4), YellowFever, Japanese encephalitis, West Nile encephalitis, St. Louisencephalitis, Russian spring-summer encephalitis, Powassan encephalitis.Flavivirus antigens may be selected from PrM, M, C, E, NS-1, NS-2a,NS2b, NS3, NS4a, NS4b, and NS5. Flavivirus antigens are preferablyselected from PrM, M and E. Commercially available TBE vaccine includeinactivated virus vaccines.

Pestivirus: Viral antigens may be derived from a Pestivirus, such asBovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Borderdisease (BDV).

Hepadnavirus: Viral antigens may be derived from a Hepadnavirus, such asHepatitis B virus. Hepadnavirus antigens may be selected from surfaceantigens (L, M and S), core antigens (HBc, HBe). Commercially availableHBV vaccines include subunit vaccines comprising the surface antigen Sprotein.

Hepatitis C virus: Viral antigens may be derived from a Hepatitis Cvirus (HCV). HCV antigens may be selected from one or more of E1, E2,E1/E2, NS345 polyprotein, NS 345-core polyprotein, core, and/or peptidesfrom the nonstructural regions (Houghton et al., Hepatology (1991)14:381).

Rhabdovirus: Viral antigens may be derived from a Rhabdovirus, such as aLyssavirus (Rabies virus) and Vesiculovirus (VSV). Rhabdovirus antigensmay be selected from glycoprotein (G), nucleoprotein (N), large protein(L), nonstructural proteins (NS). Commercially available Rabies virusvaccine comprise killed virus grown on human diploid cells or fetalrhesus lung cells.

Caliciviridae; Viral antigens may be derived from Calciviridae, such asNorwalk virus, and Norwalk-like Viruses, such as Hawaii Virus and SnowMountain Virus.

Coronavirus: Viral antigens may be derived from a Coronavirus, SARS,Human respiratory coronavirus, Avian infectious bronchitis (IBV), Mousehepatitis virus (MHV), and Porcine transmissible gastroenteritis virus(TGEV). Coronavirus antigens may be selected from spike (S), envelope(E), matrix (M), nucleocapsid (N), and Hemagglutinin-esteraseglycoprotein (HE). Preferably, the Coronavirus antigen is derived from aSARS virus. SARS viral antigens are described in WO 04/92360;

Retrovirus: Viral antigens may be derived from a Retrovirus, such as anOncovirus, a Lentivirus or a Spumavirus. Oncovirus antigens may bederived from HTLV-1, HTLV-2 or HTLV-5. Lentivirus antigens may bederived from HIV-1 or HIV-2. Retrovirus antigens may be selected fromgag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr. HIV antigensmay be selected from gag (p24gag and p55gag), env (gp160 and gp41), pol,tat, nef, rev vpu, miniproteins, (preferably p55 gag and gp140v delete).HIV antigens may be derived from one or more of the following strains:HIV_(IIIb), HIV_(SF2), HIV_(LAV), HIV_(LA1), HIV_(MN), HIV-1_(CM235),HIV-1_(US4).

Reovirus: Viral antigens may be derived from a Reovirus, such as anOrthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus. Reovirusantigens may be selected from structural proteins λ1, λ2, λ3, μ1, μ2,σ1, σ2, or σ3, or nonstructural proteins σNS, μNS, or σ1s. PreferredReovirus antigens may be derived from a Rotavirus. Rotavirus antigensmay be selected from VP1, VP2, VP3, VP4 (or the cleaved product VP5 andVP8), NSP 1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5. Preferred Rotavirusantigens include VP4 (or the cleaved product VP5 and VP8), and VP7. See,e.g., WO 2005/021033, WO 2003/072716, WO 2002/11540, WO 2001/12797, WO01/08495, WO 00/26380, WO 02/036172; herein incorporated by reference intheir entireties.

Parvovirus: Viral antigens may be derived from a Parvovirus, such asParvovirus B19. Parvovirus antigens may be selected from VP-1, VP-2,VP-3, NS-1 and NS-2. Preferably, the Parvovirus antigen is capsidprotein VP-2.

Delta hepatitis virus (HDV): Viral antigens may be derived HDV,particularly 8-antigen from HDV (see, e.g., U.S. Pat. No. 5,378,814).

Hepatitis E-virus (HEV): Viral antigens may be derived from HEV.

Hepatitis G virus (HGV): Viral antigens may be derived from HGV.

Human Herpesvirus: Viral antigens may be derived from a HumanHerpesvirus, such as Herpes Simplex Viruses (HSV), Varicella-zostervirus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), HumanHerpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus8 (HHV8). Human Herpesvirus antigens may be selected from immediateearly proteins (α), early proteins (β), and late proteins (γ). HSVantigens may be derived from HSV-1 or HSV-2 strains. HSV antigens may beselected from glycoproteins gB, gC, gD and gH, fusion protein (gB), orimmune escape proteins (gC, gE, or gI). VZV antigens may be selectedfrom core, nucleocapsid, tegument, or envelope proteins. A liveattenuated VZV vaccine is commercially available. EBV antigens may beselected from early antigen (EA) proteins, viral capsid antigen (VCA),and glycoproteins of the membrane antigen (MA). CMV antigens may beselected from capsid proteins, envelope glycoproteins (such as gB andgH), and tegument proteins

Papovaviruses: Antigens may be derived from Papovaviruses, such asPapillomaviruses and Polyomaviruses. Papillomaviruses include HPVserotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47,51, 57, 58, 63 and 65. Preferably, HPV antigens are derived fromserotypes 6, 11, 16 or 18. HPV antigens may be selected from capsidproteins (L1) and (L2), or E1-E7, or fusions thereof HPV antigens arepreferably formulated into virus-like particles (VLPs). Polyomyavirusviruses include BK virus and JK virus. Polyomavirus antigens may beselected from VP1, VP2 or VP3.

Further provided are antigens, compositions, methods, and microbesincluded in Vaccines, 4^(th) Edition (Plotkin and Orenstein ed. 2004);Medical Microbiology 4^(th) Edition (Murray et al. ed. 2002); Virology,3rd Edition (W. K. Joklik ed. 1988); Fundamental Virology, 2nd Edition(B. N. Fields and D. M. Knipe, eds. 1991), which are contemplated inconjunction with the compositions of the present invention.

C. FUNGAL ANTIGENS

Fungal antigens for use in the invention may be derived from one or moreof the fungi set forth below.

Fungal antigens may be derived from Dermatophytres, including:Epidermophyton floccusum, Microsporum audouini, Microsporum canis,Microsporum distortum, Microsporum equinum, Microsporum gypsum,Microsporum nanum, Trichophyton concentricum, Trichophyton equinum,Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini,Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophytonrubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophytonverrucosum, T. verrucosum var. album, var. discoides, var. ochraceum,Trichophyton violaceum, and/or Trichophyton faviforme.

Fungal pathogens may be derived from Aspergillus fumigatus, Aspergillusflavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus,Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus,Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candidatropicalis, Candida glabrata, Candida krusei, Candida parapsilosis,Candida stellatoidea, Candida kusei, Candida parakwsei, Candidalusitaniae, Candida pseudotropicalis, Candida guilliermondi,Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis,Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum,Klebsiella pneumoniae, Paracoccidioides brasiliensis, Pneumocystiscarinii, Pythiumn insidiosum, Pityrosporum ovale, Sacharomycescerevisae, Saccharomyces boulardii, Saccharomyces pombe, Scedosporiumapiosperum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasmagondii, Penicillium marneffei, Malassezia spp., Fonsecaea spp.,Wangiella spp., Sporothrix spp., Basidiobolus spp., Conidiobolus spp.,Rhizopus spp, Mucor spp, Absidia spp, Mortierella spp, Cunninghamellaspp, Saksenaea spp., Alternaria spp, Curvularia spp, Helminthosporiumspp, Fusarium spp, Aspergillus spp, Penicillium spp, Monolinia spp,Rhizoctonia spp, Paecilomyces spp, Pithomyces spp, and Cladosporium spp.

Processes for producing a fungal antigens are well known in the art (seeU.S. Pat. No. 6,333,164). In a preferred method a solubilized fractionextracted and separated from an insoluble fraction obtainable fromfungal cells of which cell wall has been substantially removed or atleast partially removed, characterized in that the process comprises thesteps of: obtaining living fungal cells; obtaining fungal cells of whichcell wall has been substantially removed or at least partially removed;bursting the fungal cells of which cell wall has been substantiallyremoved or at least partially removed; obtaining an insoluble fraction;and extracting and separating a solubilized fraction from the insolublefraction.

D. STD ANTIGENS

The compositions of the invention may include one or more antigensderived from a sexually transmitted disease (STD). Such antigens mayprovide for prophylactis or therapy for STD's such as chlamydia, genitalherpes, hepatitis (such as HCV), genital warts, gonorrhoea, syphilisand/or chancroid (See, WO00/15255). Antigens may be derived from one ormore viral or bacterial STD's. Viral STD antigens for use in theinvention may be derived from, for example, HIV, herpes simplex virus(HSV-1 and HSV-2), human papillomavirus (HPV), and hepatitis (HCV).Bacterial STD antigens for use in the invention may be derived from, forexample, Neiserria gonorrhoeae, Chlamydia trachomatis, Treponemapallidum, Haemophilus ducreyi, E. coli, and Streptococcus agalactiae.Examples of specific antigens derived from these pathogens are describedabove.

E. RESPIRATORY ANTIGENS

The compositions of the invention may include one or more antigensderived from a pathogen which causes respiratory disease. For example,respiratory antigens may be derived from a respiratory virus such asOrthomyxoviruses (influenza), Pneumovirus (RSV), Paramyxovirus (Hy),Morbillivirus (measles), Togavirus (Rubella), VZV, and Coronavirus(SARS). Respiratory antigens may be derived from a bacteria which causesrespiratory disease, such as Streptococcus pneumoniae, Pseudomonasaeruginosa, Bordetella pertussis, Mycobacterium tuberculosis, Mycoplasmapneumoniae, Chlamydia pneumoniae, Bacillus anthracis, and Moraxellacatarrhalis. Examples of specific antigens derived from these pathogensare described above.

F. PEDIATRIC VACCINE ANTIGENS

The compositions of the invention may include one or more antigenssuitable for use in pediatric subjects. Pediatric subjects are typicallyless than about 3 years old, or less than about 2 years old, or lessthan about 1 years old. Pediatric antigens may be administered multipletimes over the course of 6 months, 1, 2 or 3 years. Pediatric antigensmay be derived from a virus which may target pediatric populationsand/or a virus from which pediatric populations are susceptible toinfection. Pediatric viral antigens include antigens derived from one ormore of Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus(PIV and Mumps), Morbillivirus (measles), Togavirus (Rubella),Enterovirus (polio), HBV, Coronavirus (SARS), and Varicella-zoster virus(VZV), Epstein Barr virus (EBV). Pediatric bacterial antigens includeantigens derived from one or more of Streptococcus pneumoniae, Neisseriameningitides, Streptococcus pyogenes (Group A Streptococcus), Moraxellacatarrhalis, Bordetella pertussis, Staphylococcus aureus, Clostridiumtetani (Tetanus), Cornynebacterium diphtheriae (Diphtheria), Haemophilusinfluenzae B (Hib), Pseudomonas aeruginosa, Streptococcus agalactiae(Group B Streptococcus), and E. coli. Examples of specific antigensderived from these pathogens are described above.

G. ANTIGENS SUITABLE FOR USE IN ELDERLY OR IMMUNOCOMPROMISED INDIVIDUALS

The compositions of the invention may include one or more antigenssuitable for use in elderly or immunocompromised individuals. Suchindividuals may need to be vaccinated more frequently, with higher dosesor with adjuvanted formulations to improve their-immune response to thetargeted antigens. Antigens which may be targeted for use in elderly orimmunocompromised individuals include antigens derived from one or moreof the following pathogens: Neisseria meningitides, Streptococcuspneumoniae, Streptococcus pyogenes (Group A Streptococcus), Moraxellacatarrhalis, Bordetella pertussis, Staphylococcus aureus, Staphylococcusepidermis, Clostridium tetani (Tetanus), Cornynebacterium diphtheriae(Diphtheria), Haemophilus influenzae B (Hib), Pseudomonas aeruginosa,Legionella pneumophila, Streptococcus agalactiae (Group BStreptococcus), Enterococcus faecalis, Helicobacter pylori, Clamydiapneumoniae, Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus(PTV and Mumps), Morbillivirus (measles), Togavirus (Rubella),Enterovirus (polio), HBV, Coronavirus (SARS), Varicella-zoster virus(VZV), Epstein Barr virus (EBV), Cytomegalovirus (CMV). Examples ofspecific antigens derived from these pathogens are described above.

H. ANTIGENS SUITABLE FOR USE IN ADOLESCENT VACCINES

The compositions of the invention may include one or more antigenssuitable for use in adolescent subjects. Adolescents may be in need of aboost of a previously administered pediatric antigen. Pediatric antigenswhich may be suitable for use in adolescents are described above. Inaddition, adolescents may be targeted to receive antigens derived froman STD pathogen in order to ensure protective or therapeutic immunitybefore the beginning of sexual activity. STD antigens which may besuitable for use in adolescents are described above.

I. ANTIGEN FORMULATIONS

In other aspects of the invention, methods of producing microparticleshaving adsorbed antigens are provided. The methods comprise: (a)providing an emulsion by dispersing a mixture comprising (i) water, (ii)a detergent, (iii) an organic solvent, and (iv) a biodegradable polymerselected from the group consisting of a poly(α-hydroxy acid), apolyhydroxy butyric acid, a polycaprolactone, a polyorthoester, apolyanhydride, and a polycyanoacrylate. The polymer is typically presentin the mixture at a concentration of about 1% to about 30% relative tothe organic solvent, while the detergent is typically present in themixture at a weight-to-weight detergent-to-polymer ratio of from about0.00001:1 to about 0.1:1 (more typically about 0.0001:1 to about 0.1:1,about 0.001:1 to about 0.1:1, or about 0.005:1 to about 0.1:1); (b)removing the organic solvent from the emulsion; and (c) adsorbing anantigen on the surface of the microparticles. In certain embodiments,the biodegradable polymer is present at a concentration of about 3% toabout 10% relative to the organic solvent.

Microparticles for use herein will be formed from materials that aresterilizable, non-toxic and biodegradable. Such materials include,without limitation, poly(α-hydroxy acid), polyhydroxybutyric acid,polycaprolactone, polyorthoester, polyanhydride, PACA, andpolycyanoacrylate. Preferably, microparticles for use with the presentinvention are derived from a poly(α-hydroxy acid), in particular, from apoly(lactide) (“PLA”) or a copolymer of D,L-lactide and glycolide orglycolic acid, such as a poly(D,L-lactide-co-glycolide) (“PLG” or“PLGA”), or a copolymer of D,L-lactide and caprolactone. Themicroparticles may be derived from any of various polymeric startingmaterials which have a variety of molecular weights and, in the case ofthe copolymers such as PLG, a variety of lactide:glycolide ratios, theselection of which will be largely a matter of choice, depending in parton the coadministered macromolecule. These parameters are discussed morefully below.

Further antigens may also include an outer membrane vesicle (OMV)preparation. Additional formulation methods and antigens (especiallytumor antigens) are provided in U.S. patent Ser. No. 09/581,772.

J. ANTIGEN REFERENCES

The following references include antigens useful in conjunction with thecompositions of the present invention:

-   1 International patent application WO99/24578-   2 International patent application WO99/36544.-   3 International patent application WO99/57280.-   4 International patent application WO00/22430.-   5 Tettelin et al. (2000) Science 287:1809-1815.-   6 International patent application WO96/29412.-   7 Pizza et al. (2000) Science 287:1816-1820.-   8 PCT WO 01/52885.-   9 Bjune et al. (1991) Lancet 338(8775).-   10 Fuskasawa et al. (1999) Vaccine 17:2951-2958.-   11 Rosenqist et al. (1998) Dev. Biol. Strand 92:323-333.-   12 Constantino et al. (1992) Vaccine 10:691-698.-   13 Constantino et al. (1999) Vaccine 17:1251-1263.-   14 Watson (2000) Pediatr Infect Dis J 19:331-332.-   15 Rubin (20000) Pediatr Clin North Am 47:269-285, v.-   16 Jedrzeja's (2001) Microbiol Mol Biol Rev 65:187-207.-   17 International patent application filed on 3^(rd) Jul. 2001    claiming priority from GB-0016363.4; WO 02/02606; PCT IB/01/00166.-   18 Kalman et al. (1999) Nature Genetics 21:385-389.-   19 Read et al. (2000) Nucleic Acids Res 28:1397-406.-   20 Shirai et al. (2000) J. Infect. Dis 181(Suppl 3):S524-5527.-   21 International patent application WO99/27105.-   22 International patent application WO00/27994.-   23 International patent application WO00/37494.-   24 International patent application WO99/28475.-   25 Bell (2000) Pediatr Infect Dis J 19:1187-1188.-   26 Iwarson (1995) APMIS103:321-326.-   27 Gerlich et al. (1990) Vaccine 8 Suppl:S63-68 & 79-80.-   28 Hsu et al. (1999) Clin Liver Dis 3:901-915.-   29 Gastofsson et al. (1996) N. Engl. J. Med. 334-:349-355.-   30 Rappuoli et al. (1991) TIBTECH 9:232-238.-   31 Vaccines (1988) eds. Plotkin & Mortimer. ISBN 0-7216-1946-0.-   32 Del Guidice et al. (1998) Molecular Aspects of Medicine 19:1-70.-   33 International patent application WO93/018150.-   34 International patent application WO99/53310.-   35 International patent application WO98/04702.-   36 Ross et al. (2001) Vaccine 19:135-142.-   37 Sutter et al. (2000) Pediatr Clin North Am 47:287-308.-   38 Zimmerman & Spann (1999) Am Fan Physician 59:113-118, 125-126.-   39 Dreensen (1997) Vaccine 15 Suppl” S2-6.-   40 MMWR Morb Mortal Wkly rep 1998 January 16:47(1):12, 9.-   41 McMichael (2000) Vaccine 19 Suppl 1:S101-107.-   42 Schuchat (1999) Lancer 353(9146):51-6.-   43 GB patent applications 0026333.5, 0028727.6 & 0105640.7.-   44 Dale (1999) Infect Disclin North Am 13:227-43, viii.-   45 Ferretti et al. (2001) PNAS USA 98: 4658-4663.-   46 Kuroda et al. (2001) Lancet 357(9264):1225-1240; see also pages    1218-1219.-   47 Ramsay et al. (2001) Lancet 357(9251):195-196.-   48 Lindberg (1999) Vaccine 17 Suppl 2:S28-36.-   49 Buttery & Moxon (2000) J R Coil Physicians Long 34:163-168.-   50 Ahmad & Chapnick (1999) Infect Dis Clin North Am 13:113-133, vii.-   51 Goldblatt (1998) J. Med. Microbiol. 47:663-567.-   52 European patent 0 477 508.-   53 U.S. Pat. No. 5,306,492.-   54 International patent application WO98/42721.-   55 Conjugate Vaccines (eds. Cruse et al.) ISBN 3805549326,    particularly vol. 10:48-114.-   56 Hermanson (1996) Bioconjugate Techniques ISBN: 012323368 &    012342335X.-   57 European patent application 0372501.-   58 European patent application 0378881.-   59 European patent application 0427347.-   60 International patent application WO93/17712.-   61 International patent application WO98/58668.-   62 European patent application 0471177.-   63 International patent application WO00/56360.-   64 International patent application WO00/67161.    The contents of all of the above cited patents, patent applications    and journal articles are incorporated by reference as if set forth    fully herein.

The immunogenic compositions of the invention may be prepared in variousforms. For example, the compositions may be prepared as injectables,either as liquid solutions or suspensions. Solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection canalso be prepared (e.g. a lyophilized composition or a spray-freeze driedcomposition). The composition may be prepared for topical administratione.g. as an ointment, cream or powder. The composition may be preparedfor oral administration e.g. as a tablet or capsule, as a spray, or as asyrup (optionally flavoured) and/or a fast dissolving dosage form. Thecomposition may be prepared for pulmonary administration e.g. as aninhaler, using a fine powder or a spray. The composition may be preparedas a suppository or pessary. The composition may be prepared for nasal,aural or ocular administration e.g. as drops. Preparation of suchpharmaceutical compositions is within the general skill of the art. See,e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa., 18th edition, 1990.

The composition may be in kit form, designed such that a combinedcomposition is reconstituted just prior to administration to a patient.Such kits may comprise one or more Norovirus and/or Sapovirus antigensor nucleic acids encoding such antigens in liquid form, and any of theadditional antigens and adjuvants as described herein.

Immunogenic compositions of the invention comprising polypeptideantigens or nucleic acid molecules are preferably vaccine compositions.The pH of such compositions preferably is between 6 and 8, preferablyabout 7. The pH can be maintained by the use of a buffer. Thecomposition can be sterile and/or pyrogen-free. The composition can beisotonic with respect to humans. Vaccines according to the invention maybe used either prophylactically or therapeutically, but will typicallybe prophylactic and can be used to treat animals (including companionand laboratory mammals), particularly humans.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of antigen(s) and/or nucleic acids encoding antigen(s),as well as any other components, as needed. By ‘immunologicallyeffective amount’, it is meant that the administration of that amount toan individual, either in a single dose or as part of a series, iseffective for treatment or prevention. This amount varies depending uponthe health and physical condition of the individual to be treated, age,the taxonomic group of individual to be treated (e.g. human, non-humanprimate, etc.), the capacity of the individual's immune system tosynthesize antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials.

G. Administration

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or mucosally, such as byrectal, oral (e.g. tablet, spray), vaginal, topical, transdermal (Seee.g. WO99/27961) or transcutaneous (See e.g. WO02/074244 andWO02/064162), intranasal (See e.g. WO03/028760), ocular, aural,pulmonary or other mucosal administration Immunogenic compositions canalso be administered topically by direct transfer to the surface of theskin. Topical administration can be accomplished without utilizing anydevices, or by contacting naked skin with the immunogenic compositionutilizing a bandage or a bandage-like device (see, e.g., U.S. Pat. No.6,348,450).

Preferably the mode of administration is parenteral, mucosal or acombination of mucosal and parenteral immunizations. Even morepreferably, the mode of administration is parenteral, mucosal or acombination of mucosal and parenteral immunizations in a total of 1-2vaccinations 1-3 weeks apart. Preferably the route of administrationincludes but is not limited to oral delivery, intra-muscular deliveryand a combination of oral and intra-muscular delivery.

It has already been demonstrated that mucosal and systemic immuneresponses to antigens, such as Helicobacter pylori antigens can beenhanced through mucosal priming followed by systemic boostingimmunizations (see Vajdy et al (2003) Immunology 110: 86-94). In apreferred embodiment, the method for treating an infection by aNorovirus or Sapovirus, comprises mucosally administering to a subjectin need thereof a first immunogenic composition comprising one or moreNorovirus or Sapovirus antigens followed by parenterally administering atherapeutically effective amount of a second immunogenic compositioncomprising one or more Norovirus or Sapovirus antigens.

The immunogenic composition may be used to elicit systemic and/ormucosal immunity, preferably to elicit an enhanced systemic and/ormucosal immunity.

Preferably the immune response is characterized by the induction of aserum IgG and/or intestinal IgA immune response.

As noted above, prime-boost methods are preferably employed where one ormore gene delivery vectors and/or polypeptide antigens are delivered ina “priming” step and, subsequently, one or more second gene deliveryvectors and/or polypeptide antigens are delivered in a “boosting” step.In certain embodiments, priming and boosting with one or more genedelivery vectors or polypeptide antigens described herein is followed byadditional boosting with one or more polypeptide-containing compositions(e.g., polypeptides comprising Norovirus and/or Sapovirus antigens).

In any method involving co-administration, the various compositions canbe delivered in any order. Thus, in embodiments including delivery ofmultiple different compositions or molecules, the nucleic acids need notbe all delivered before the polypeptides. For example, the priming stepmay include delivery of one or more polypeptides and the boostingcomprises delivery of one or more nucleic acids and/or one or morepolypeptides. Multiple polypeptide administrations can be followed bymultiple nucleic acid administrations or polypeptide and nucleic acidadministrations can be performed in any order. Thus, one or more of thegene delivery vectors described herein and one or more of thepolypeptides described herein can be co-administered in any order andvia any administration route. Therefore, any combination ofpolynucleotides and polypeptides described herein can be used to elicitan immune reaction.

Dosage Regime

Dosage treatment can be according to a single dose schedule or amultiple dose schedule. Multiple doses may be used in a primaryimmunization schedule and/or in a booster immunization schedule. In amultiple dose schedule, the various doses may be given by the same ordifferent routes, e.g. a parenteral prime and mucosal boost, a mucosalprime and parenteral boost, etc.

Preferably the dosage regime enhances the avidity of the antibodyresponse leading to antibodies with a neutralizing characteristic. Anin-vitro neutralization assay may be used to test for neutralizingantibodies (see for example Asanaka et al (2005) J of Virology 102:10327; Wobus et al (2004) PLOS Biology 2(12); e432; and Dubekti et al(2002) J Medical Virology 66: 400).

There is a strong case for a correlation between serum antibody levelsand protection from disease caused by Norovirus and/or Saporovirus. Forexample, in multiple challenge studies, serum antibody levels wereassociated with protection after repeated (2-3) oral challenges withhigh doses of Norwalk virus (Journal of Infectious Disease (1990)161:18). In another study, 18 of 23 infants without pre-existingantibodies developed gastroenteritis caused by human Caliciviruses,whereas 15 of 18 with pre-existing antibody levels did not become ill(Journal of Infectious Disease (1985). In yet another study, 47% ofpersons with a baseline Norwalk antibody titre of less than 1:100developed Norwalk infection compared to 13% of persons with a baselineantibody titre of greater than 1:100 (p<0.001) (Journal of InfectiousDisease (1985) 151: 99).

H. Tests to Determine the Efficacy of an Immune Response

One way of assessing efficacy of therapeutic treatment involvesmonitoring infection after administration of a composition of theinvention. One way of assessing efficacy of prophylactic treatmentinvolves monitoring immune responses against the antigens in thecompositions of the invention after administration of the composition.

Another way of assessing the immunogenicity of the component proteins ofthe immunogenic compositions of the present invention is to express theproteins recombinantly and to screen patient sera or mucosal secretionsby immunoblot. A positive reaction between the protein and the patientserum indicates that the patient has previously mounted an immuneresponse to the protein in question—that is, the protein is animmunogen. This method may also be used to identify immunodominantproteins and/or epitopes.

Another way of checking efficacy of therapeutic treatment involvesmonitoring infection after administration of the compositions of theinvention. One way of checking efficacy of prophylactic treatmentinvolves monitoring immune responses both systemically (such asmonitoring the level of IgG1 and IgG2a production) and mucosally (suchas monitoring the level of IgA production) against the antigens in thecompositions of the invention after administration of the composition.Typically, serum specific antibody responses are determinedpost-immunization but pre-challenge whereas mucosal specific antibodybody responses are determined post-immunization and post-challenge.

The immunogenic compositions of the present invention can be evaluatedin in vitro and in vivo animal models prior to host, e.g., human,administration. Particularly useful mouse models include those in whichintraperitoneal immunization is followed by either intraperitonealchallenge or intranasal challenge.

The efficacy of immunogenic compositions of the invention can also bedetermined in vivo by challenging animal models of infection, e.g.,guinea pigs or mice or rhesus macaques, with the immunogeniccompositions. The immunogenic compositions may or may not be derivedfrom the same strains as the challenge strains. Preferably theimmunogenic compositions are derivable from the same strains as thechallenge strains.

In vivo efficacy models include but are not limited to: (i) A murineinfection model using human strains; (ii) a murine disease model whichis a murine model using a mouse-adapted strain, such as strains whichare particularly virulent in mice and (iii) a primate model using humanisolates. A human challenge model, which is supported by the NIH andCenter for Disease Control (CDC) is also available (see for example,Lindesmith et al (2003) Nature Medicine 9: 548-553 and Journal ofVirology (2005) 79: 2900).

The immune response may be one or both of a TH1 immune response and aTH2 response. The immune response may be an improved or an enhanced oran altered immune response. The immune response may be one or both of asystemic and a mucosal immune response. Preferably the immune responseis an enhanced systemic and/or mucosal response.

An enhanced systemic and/or mucosal immunity is reflected in an enhancedTH1 and/or TH2 immune response. Preferably, the enhanced immune responseincludes an increase in the production of IgG1 and/or IgG2a and/or IgA.Preferably the mucosal immune response is a TH2 immune response.Preferably, the mucosal immune response includes an increase in theproduction of IgA.

Activated TH2 cells enhance antibody production and are therefore ofvalue in responding to extracellular infections. Activated TH2 cells maysecrete one or more of IL-4, IL-5, IL-6, and IL-10. A TH2 immuneresponse may result in the production of IgG1, IgE, IgA and memory Bcells for future protection.

A TH2 immune response may include one or more of an increase in one ormore of the cytokines associated with a TH2 immune response (such asIL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgG1,IgE, IgA and memory B cells. Preferably, the enhanced TH2 immuneresponse will include an increase in IgG1 production.

A TH1 immune response may include one or more of an increase in CTLs, anincrease in one or more of the cytokines associated with a TH1 immuneresponse (such as IL-2, IFNγ, and TNFβ), an increase in activatedmacrophages, an increase in NK activity, or an increase in theproduction of IgG2a. Preferably, the enhanced TH1 immune response willinclude an increase in IgG2a production.

Immunogenic compositions of the invention, in particular, immunogeniccomposition comprising one or more antigens of the present invention maybe used either alone or in combination with other antigens optionallywith an immunoregulatory agent capable of eliciting a Th1 and/or Th2response.

The invention also comprises an immunogenic composition comprising oneor more immunoregulatory agent, such as a mineral salt, such as analuminium salt and an oligonucleotide containing a CpG motif. Mostpreferably, the immunogenic composition includes both an aluminium saltand an oligonucleotide containing a CpG motif. Alternatively, theimmunogenic composition includes an ADP ribosylating toxin, such as adetoxified ADP ribosylating toxin and an oligonucleotide containing aCpG motif. Preferably, the one or more immunoregulatory agents includean adjuvant. The adjuvant may be selected from one or more of the groupconsisting of a TH1 adjuvant and TH2 adjuvant, further discussed above.

The immunogenic compositions of the invention will preferably elicitboth a cell mediated immune response as well as a humoral immuneresponse in order to effectively address an infection. This immuneresponse will preferably induce long lasting (e.g., neutralizing)antibodies and a cell mediated immunity that can quickly respond uponexposure to one or more infectious antigens. By way of example, evidenceof neutralizing antibodies in patients blood samples is considered as asurrogate parameter for protection since their formation is of decisiveimportance for virus elimination in TBE infections (see Kaiser andHolzmann (2000) Infection 28; 78-84).

I. Use of the Immunogenic Compositions as Medicaments

The invention also provides a composition of the invention for use as amedicament. The medicament is preferably able to raise an immuneresponse in a mammal (i.e. it is an immunogenic composition) and is morepreferably a vaccine. The invention also provides the use of thecompositions of the invention in the manufacture of a medicament forraising an immune response in a mammal. The medicament is preferably avaccine. Preferably the vaccine is used to prevent and/or treat anintestinal infection such as gastroenteritis, preferably acutegastroenteritis. The gastroenteritis may result from an imbalance in ionand/or water transfer resulting in both watery diarrhea and/orintestinal peristalisis and/or motility (vomiting).

The invention provides methods for inducing or increasing an immuneresponse using the compositions described above. The immune response ispreferably protective and can include antibodies and/or cell-mediatedimmunity (including systemic and mucosal immunity). Immune responsesinclude booster responses.

The invention also provides a method for raising an immune response in amammal comprising the step of administering an effective amount of acomposition of the invention. The immune response is preferablyprotective and preferably involves antibodies and/or cell-mediatedimmunity. Preferably, the immune response includes one or both of a TH1immune response and a TH2 immune response. The method may raise abooster response.

The mammal is preferably a human. Where the immunogenic composition,preferably a vaccine is for prophylactic use, the human is preferably achild (e.g. a toddler or infant, preferably pre-school, preferably oneyear or less or from three years (preferably 1-4 years) onwards) or ateenager; where the vaccine is for therapeutic use, the human ispreferably a teenager or an adult. A vaccine intended for children mayalso be administered to adults e.g. to assess safety, dosage,immunogenicity, etc. Preferably, the human is a teenager. Morepreferably, the human is a pre-adolescent teenager. Even morepreferably, the human is a pre-adolescent female or male. Preferably thepre-adolescent male or female is around 9-12 years of age. Preferablythe adolescent male or female is around 15-19 years of age. Preferablythe male or female is around 20-49 years of age. Preferably the male orfemale is over 49 years of age. Preferably the human is elderly,preferably around 60-80 years of age.

Other target groups for the immunogenic compositions (e.g., vaccines) ofthe present invention include: transplant and immunocompromisedindividuals; Adults and children in USA, Canada and Europe including butnot limited to the following:

Food handlers;

Healthcare workers such as but not limited to Hospital and Nursing homepersonnel;

Day care children;

Travelers including cruise ship travelers;

Military personnel; and

Paediatric and/or elderly populations as discussed above.

J. Kits

The invention also provides kits comprising one or more containers ofcompositions of the invention. Compositions can be in liquid form or canbe lyophilized, as can individual antigens. Suitable containers for thecompositions include, for example, bottles, vials, syringes, and testtubes. Containers can be formed from a variety of materials, includingglass or plastic. A container may have a sterile access port (forexample, the container may be an intravenous solution bag or a vialhaving a stopper pierceable by a hypodermic injection needle).

The kit can further comprise a second container comprising apharmaceutically-acceptable buffer, such as phosphate-buffered saline,Ringer's solution, or dextrose solution. It can also contain othermaterials useful to the end-user, including other pharmaceuticallyacceptable formulating solutions such as buffers, diluents, filters,needles, and syringes or other delivery device. The kit may furtherinclude a third component comprising an adjuvant.

The kit can also comprise a package insert containing writteninstructions for methods of inducing immunity or for treatinginfections. The package insert can be an unapproved draft package insertor can be a package insert approved by the Food and Drug Administration(FDA) or other regulatory body.

The invention also provides a delivery device pre-filled with theimmunogenic compositions of the invention.

K. Methods of Producing Norovirus or Sapovirus-Specific Antibodies

The Norovirus and Sapovirus polypeptides described herein can be used toproduce Norovirus or Sapovirus-specific polyclonal and monoclonalantibodies that specifically bind to Norovirus or Sapovirus antigens,respectively. Polyclonal antibodies can be produced by administering aNorovirus or Sapovirus polypeptide to a mammal, such as a mouse, arabbit, a goat, or a horse. Serum from the immunized animal is collectedand the antibodies are purified from the plasma by, for example,precipitation with ammonium sulfate, followed by chromatography,preferably affinity chromatography. Techniques for producing andprocessing polyclonal antisera are known in the art.

Monoclonal antibodies directed against Norovirus or Sapovirus-specificepitopes present in the polypeptides can also be readily produced.Normal B cells from a mammal, such as a mouse, immunized with aNorovirus or Sapovirus polypeptide, can be fused with, for example,HAT-sensitive mouse myeloma cells to produce hybridomas. Hybridomasproducing Norovirus or Sapovirus-specific antibodies can be identifiedusing RIA or ELISA and isolated by cloning in semi-solid agar or bylimiting dilution. Clones producing Norovirus or Sapovirus-specificantibodies are isolated by another round of screening.

Antibodies, either monoclonal and polyclonal, which are directed againstNorovirus or Sapovirus epitopes, are particularly useful for detectingthe presence of Norovirus or Sapovirus antigens in a sample, such as aserum sample from a Norovirus or Sapovirus-infected human. Animmunoassay for a Norovirus or Sapovirus antigen may utilize oneantibody or several antibodies. An immunoassay for a Norovirus orSapovirus antigen may use, for example, a monoclonal antibody directedtowards a Norovirus or Sapovirus epitope, a combination of monoclonalantibodies directed towards epitopes of one Norovirus or Sapoviruspolypeptide, monoclonal antibodies directed towards epitopes ofdifferent Norovirus or Sapovirus polypeptides, polyclonal antibodiesdirected towards the same Norovirus or Sapovirus antigen, polyclonalantibodies directed towards different Norovirus or Sapovirus antigens,or a combination of monoclonal and polyclonal antibodies. Immunoassayprotocols may be based, for example, upon competition, direct reaction,or sandwich type assays using, for example, labeled antibody. The labelsmay be, for example, fluorescent, chemiluminescent, or radioactive.

The polyclonal or monoclonal antibodies may further be used to isolateNorovirus or Sapovirus particles or antigens by immunoaffinity columns.The antibodies can be affixed to a solid support by, for example,adsorption or by covalent linkage so that the antibodies retain theirimmunoselective activity. Optionally, spacer groups may be included sothat the antigen binding site of the antibody remains accessible. Theimmobilized antibodies can then be used to bind Norovirus or Sapovirusparticles or antigens from a biological sample, such as blood or plasma.The bound Norovirus or Sapovirus particles or antigens are recoveredfrom the column matrix by, for example, a change in pH.

L. Norovirus and Sapovirus Specific T Cells

Norovirus or Sapovirus-specific T cells, which are activated by theabove-described immunogenic polypeptides, polyproteins, multiepitopefusion proteins, or VLPs expressed in vivo or in vitro, preferablyrecognize an epitope of a Norovirus or Sapovirus polypeptide, such as aVP1 or VP2 polypeptide or a nonstructural polypeptide. Norovirus orSapovirus-specific T cells can be CD8⁺ or CD4⁺.

Norovirus or Sapovirus-specific CD8⁺ T cells can be cytotoxic Tlymphocytes (CTL) which can kill Norovirus or Sapovirus-infected cellsthat display any of these epitopes complexed with an MHC class Imolecule. Norovirus or Sapovirus-specific CD8⁺ T cells can be detectedby, for example, ⁵¹Cr release assays (see Example 4). ⁵¹Cr releaseassays measure the ability of Norovirus or Sapovirus-specific CD8⁺ Tcells to lyse target cells displaying one or more of these epitopes.Norovirus or Sapovirus-specific CD8⁺ T cells which express antiviralagents, such as IFN-γ, are also contemplated herein and can also bedetected by immunological methods, preferably by intracellular stainingfor IFN-γ or like cytokine after in vitro stimulation with one or moreof the Norovirus or Sapovirus polypeptides, such as but not limited to aVP1, VP2, VP10, or nonstructural polypeptide, (see Example 5).

Norovirus or Sapovirus-specific CD4⁺ T cells can be detected by alymphoproliferation assay (see Example 6). Lymphoproliferation assaysmeasure the ability of Norovirus or Sapovirus-specific CD4⁺ T cells toproliferate in response to, e.g., a VP1, VP2, VP10, and/or anonstructural polypeptide epitope.

Methods of Activating Norovirus or Sapovirus-Specific T Cells

The Norovirus or Sapovirus polynucleotides and/or immunogenicpolypeptides, polyproteins, and/or multiepitope fusion proteins can beused to activate Norovirus or Sapovirus-specific T cells either in vitroor in vivo. Activation of Norovirus or Sapovirus-specific T cells can beused, inter alia, to provide model systems to optimize CTL responses toNorovirus or Sapovirus and to provide prophylactic or therapeutictreatment against Norovirus or Sapovirus infection. For in vitroactivation, proteins are preferably supplied to T cells via a plasmid ora viral vector, such as an adenovirus vector, as described above.

Polyclonal populations of T cells can be derived from the blood, andpreferably from peripheral lymphoid organs, such as lymph nodes, spleen,or thymus, of mammals that have been infected with a Norovirus orSapovirus. Preferred mammals include mice, chimpanzees, baboons, andhumans. Infection with Norovirus or Sapovirus serves to expand thenumber of activated Norovirus or Sapovirus-specific T cells in themammal. The Norovirus or Sapovirus-specific T cells derived from themammal can then be restimulated in vitro by adding, a Norovirus orSapovirus immunogenic polypeptide, polyprotein, and/or multiepitopefusion protein. The Norovirus or Sapovirus-specific T cells can then betested for, inter alia, proliferation, the production of IFN-γ, and theability to lyse target cells displaying, for example, VP1, VP2, VP10, ornonstructural polypeptide epitopes in vitro.

In a lymphoproliferation assay (see Example 6), Norovirus orSapovirus-activated CD4⁺ T cells proliferate when cultured with aNorovirus or Sapovirus immunogenic polypeptide, polyprotein, and/ormultiepitope fusion protein, but not in the absence of such animmunogenic polypeptide. Thus, particular Norovirus or Sapovirusepitopes, such as derived from VP1, VP2, VP10, and nonstructuralpolypeptides, and fusions of these epitopes that are recognized byNorovirus or Sapovirus-specific CD4⁺ T cells can be identified using alymphoproliferation assay.

Similarly, detection of IFN-γ in Norovirus or Sapovirus-specific CD4+and/or CD8⁺ T cells after in vitro stimulation with the above-describedimmunogenic polypeptides, can be used to identify, for example,epitopes, such as but not limited to VP1, VP2, VP10, and nonstructuralpolypeptides, and fusions of these epitopes that are particularlyeffective at stimulating CD4+ and/or CD8⁺ T cells to produce IFN-γ (seeExample 5).

Further, ⁵¹Cr release assays are useful for determining the level of CTLresponse to Norovirus or Sapovirus. See Cooper et al. Immunity10:439-449. For example, Norovirus or Sapovirus-specific CD8⁺ T cellscan be derived from the liver of an Norovirus or Sapovirus infectedmammal. These T cells can be tested in ⁵¹Cr release assays againsttarget cells displaying, e.g., VP1, VP2, VP10, and nonstructuralpolypeptides epitopes. Several target cell populations expressingdifferent VP1, VP2, VP10, and nonstructural polypeptides epitopes can beconstructed so that each target cell population displays differentepitopes of VP1, VP2, VP10, and nonstructural polypeptides. TheNorovirus or Sapovirus-specific CD8⁺ cells can be assayed against eachof these target cell populations. The results of the ⁵¹Cr release assayscan be used to determine which epitopes of VP1, VP2, VP10, andnonstructural polypeptides are responsible for the strongest CTLresponse to Norovirus or Sapovirus.

Norovirus or Sapovirus immunogenic polypeptides, polyproteins,multiepitope fusion proteins, and/or VLPs as described above, and/orpolynucleotides encoding such polypeptides, can be administered to amammal, such as a mouse, baboon, chimpanzee, or human, to activateNorovirus or Sapovirus-specific T cells in vivo. Administration can beby any means known in the art, including parenteral, intranasal,intramuscular or subcutaneous injection, including injection using abiological ballistic gun (“gene gun”), as discussed above.

Preferably, injection of a Norovirus or Sapovirus polynucleotide is usedto activate T cells. In addition to the practical advantages ofsimplicity of construction and modification, injection of thepolynucleotides results in the synthesis of immunogenic polypeptide inthe host. Thus, these immunogens are presented to the host immune systemwith native post-translational modifications, structure, andconformation. The polynucleotides are preferably injectedintramuscularly to a large mammal, such as a human, at a dose of 0.5,0.75, 1.0, 1.5, 2.0, 2.5, 5 or 10 mg/kg.

A composition of the invention comprising a Norovirus or Sapovirusimmunogenic polypeptide, VLP, or polynucleotide is administered in amanner compatible with the particular composition used and in an amountwhich is effective to activate Norovirus or Sapovirus-specific T cellsas measured by, inter alia, a ⁵¹Cr release assay, a lymphoproliferationassay, or by intracellular staining for IFN-γ. The proteins and/orpolynucleotides can be administered either to a mammal which is notinfected with a Norovirus or Sapovirus or can be administered to aNorovirus or Sapovirus-infected mammal. The particular dosages of thepolynucleotides or fusion proteins in a composition will depend on manyfactors including, but not limited to the species, age, and generalcondition of the mammal to which the composition is administered, andthe mode of administration of the composition. An effective amount ofthe composition of the invention can be readily determined using onlyroutine experimentation. In vitro and in vivo models described above canbe employed to identify appropriate doses. The amount of polynucleotideused in the example described below provides general guidance which canbe used to optimize the activation of Norovirus or Sapovirus-specific Tcells either in vivo or in vitro. Generally, 0.5, 0.75, 1.0, 1.5, 2.0,2.5, 5 or 10 mg of a Norovirus or Sapovirus polypeptide orpolynucleotide, will be administered to a large mammal, such as ababoon, chimpanzee, or human. If desired, co-stimulatory molecules oradjuvants can also be provided before, after, or together with thecompositions.

Immune responses of the mammal generated by the delivery of acomposition of the invention, including activation of Norovirus orSapovirus-specific T cells, can be enhanced by varying the dosage, routeof administration, or boosting regimens. Compositions of the inventionmay be given in a single dose schedule, or preferably in a multiple doseschedule in which a primary course of vaccination includes 1-10 separatedoses, followed by other doses given at subsequent time intervalsrequired to maintain and/or reinforce an immune response, for example,at 1-4 months for a second dose, and if needed, a subsequent dose ordoses after several months.

III. EXPERIMENTAL

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperatures, etc.), but some experimental error anddeviation should, of course, be allowed for.

Example 1 Expression of Norwalk Virus Capsid Protein in Yeast

Constructs for production of Norwalk virus (NV) VLPs in Saccharomycescerevisiae were created by cloning sequences encoding viral capsidproteins into the yeast expression vector pBS24.1. The pBS24.1 vector isdescribed in detail in commonly owned U.S. patent application Ser. No.382,805, filed Jul. 19, 1989, which application is hereby incorporatedby reference in its entirety herein. The pBS24.1 vector contains the 2μsequence for autonomous replication in yeast and the yeast genes leu2dand URA3 as selectable markers. The β-lactamase gene and the ColE1origin of replication, required for plasmid replication in bacteria, arealso present in this expression vector. Regulation of expression was putunder the control of a hybrid ADH2/GAPDH promoter (described in U.S.Pat. No. 6,183,985) and an alpha-factor terminator.

The constructs created and utilized for expression of NV capsid proteinsincluded: NV.orf2 comprising a modified polynucleotide sequence of orf2(SEQ ID NO:1) and NV.orf2+3 comprising modified polynucleotide sequencesof orf2 and orf3 (SEQ ID NO:2). The coding sequences for orf2 (majorcapsid gene) and orf2+3 were generated using synthetic oligonucleotides,based on the DNA sequence from GenBank accession number M87661. A numberof silent mutations were introduced into orf2 and orf3 to facilitate thecloning of NV.orf2 and NV.orf2+3 in the expression vector (FIG. 1).

The full-length orf2+3 coding and 3′UTR sequence was divided into fourdomains as follows (FIG. 2):

Domain 1 (“5p”) encodes a 5′ HindIII cloning site followed by thesequence ACAAAACAAA (SEQ ID NO:27), the initiator ATG, and the first 154amino acids of the capsid protein, ending with a unique XbaI cloningsite.

Domain 2 (“mid”) encodes the next 175 amino acids, from the XbaI site toa unique AseI cloning site.

Domain 3 (“3p”) encodes the final 200 amino acids for orf2, from AseI toa unique Bspe1 site near the end of the orf2 coding sequence, thenfollowed by two stop codons and a SalI cloning site.

Domain 4 (“orf3”) includes the following: a unique BspE1 site, a stopcodon, a frame-shift/reinitiation codon that subsequently begins thetranslation of orf3 (212 amino acids), 66 bp of 3′ UTR, and finally aSalI cloning site.

The oligonucleotides for each domain were engineered to include EcoR1and SalI sites at the 5′ and 3′ ends, flanking the unique cloning sitesdescribed above. Then the kinased, annealed oligos for each domain wereligated into a pUC19 EcoR1/SalI subcloning vector (FIG. 3). Aftertransformation into HB101 competent cells (commercially available),miniscreen analysis and sequence verification, the clones with thecorrect sequence were identified as follows and amplified:

pUC19.NV.5p #4

pUC19.NV.mid #11 and #13

pUC19.NV.3p #22

pUC19.NV.orf3 #31

To assemble the full-length NV.orf2 as a HindIII/SalI fragment, a seriesof digests were performed: pUC19.NV.5p #13 was digested with HindIII andXbaI to isolate a 478 bp fragment; pUC19.NV.mid #13 was digested withXbaI and PciI to isolate a 393 bp fragment; pUC19.NV.mid #11 wasdigested with PciI and AseI to isolate a 133 bp fragment; andpUC19.NV.3p #22 was digested with AseI and SalI to isolate a 609 bpfragment. All four fragments were gel purified and ligated into thepSP72 HindIII/SalI vector, to create a 1613 bp HindIII-SalI insert forthe coding sequence of NV.orf2 (FIGS. 3 and 4).

The full-length NV.orf2+3 coding sequence was assembled by ligating theHindIII/XbaI, XbaI/PciI, and PciI/AseI fragments (described above) witha 595 bp gel purified fragment obtained from digesting pUC19.NV.3p #22with AseI and BspE1, and a gel purified BspEI/SalI fragment of 715 bp,obtained from pUC19.NV.orf3 #31, into the pSP72 HindIII/SalI vector(FIG. 5). After transformation into HB101 and miniscreen analysis, thefull-length subclones pSP72.NV.orf2 #1 and pSP72.NV.orf2+3 #16 wereobtained. The 1613 bp HindIII/SalI NV.orf2 fragment and the 2314 bpNV.orf2+3 fragment were gel isolated and purified after restrictiondigestion of the respective pSP72 subclones. Each HindIII-SalI fragmentwas ligated with the BamHI/HindIII ADH2/GAPDH yeast hybrid promoter of1366 bp into the pBS24.1 BamHI/SalI yeast expression vector, containingthe elements described above. After HB101 transformation and miniscreenanalysis, the following yeast expression plasmids were identified andamplified: pd.NV.orf2 #1 and pd.NV.orf2+3 #12 (FIGS. 6 and 7).

S. cerevisiae strain AD3 [matα, leu2Δ, trp1, ura3-52, prb-1122, pep4-3,prc1-407, cir°, trp+, ::DM15[GAP/ADR] was transformed with theexpression plasmids pd.NV.orf2 #1 and pd.NV.orf2+3 #12 using a lithiumacetate protocol (Invitrogen EasyComp). After transformation, severalUra-transformants were streaked onto Ura-8% glucose plates in order toobtain single colonies. The single colonies were subsequently patchedonto Leu-8% glucose plates to increase the plasmid copy number.Leu-starter cultures were grown for 24 hours at 30° C. and then diluted1:20 in YEPD (yeast extract bactopeptone 2% glucose) media. Cells weregrown for 48 hours at 30° C. to allow depletion of the glucose in themedia and then harvested. Then aliquots of the yeast cells were lysedwith glass beads in lysis buffer (10 mM NaPO4 pH7.5 0.1% Triton X-100).The lysates were cleared by centrifugation in 4° microfuge. Therecombinant proteins were detected in the cleared glass bead lysateusing the commercially available RIDASCREEN Norovirus Immunoassay(SciMedx Corporation) (FIG. 8). The lysates were subjected to sucrosegradient sedimentation, and the fractions were assayed using theNorovirus kit to determine if the expression of the capsid protein in S.cerevisiae resulted in the self-assembly of recombinant NV emptyvirus-like particles. Preliminary results of electron microscopyindicated the formation of virus-like particles in the peak fractions ofthe sucrose gradients (FIG. 9).

Example 2 Expression of Norwalk Virus Capsid Protein in Insect Cells

For the expression of NV capsid orf2 and NV capsid orf2+3 in the insectcell system, the following manipulations were undertaken to create anNheI/SalI fragment that could be cloned into PBLUEBAC4.5 baculovirusexpression vector. First, the 5′ end of the orf2 and orf2+3 HindIII/SalIfragments were modified to replace the HindIII restriction site with aNheI restriction site. This was accomplished with a 63 bp syntheticoligo that included the NheI site at the beginning, a sequence encodingamino acids 1-21 of the capsid protein, and a KpnI site at the end.Next, a 1534 bp KpnI/SalI NV.orf2 fragment and a 2235 bp KpnI/SalINV.orf2+3 fragment were isolated by digesting pSP72.NV.orf2 #1 andpSP72.NV.orf2+3 #16, respectively, with KpnI and SalI followed by gelelectrophoretic separation and purification of the isolated bands. TheNheI/KpnI oligos and the KpnI/SalI fragments were ligated into thePCET906A shuttle vector (ML Labs). Competent HB101 were transformed withthe ligation mixture and plated onto Luria-ampicillin plates. Afterminiprep analysis, identification of the desired clones, and sequenceconfirmation, the plasmids pCET906A.TPA_(L).orf2 #21 andpCET906A.TPA_(L).orf2+3 #34 were amplified (FIG. 10).

Next pCET906A.TPA_(L).orf2 #21 and pCET906A.TPA_(L).orf2+3 #34 weredigested with NheI and SalI to gel isolate a 1602 bp fragment coding forNV.orf2 and a 2303 bp fragment coding for NV.orf2+3, respectively. Eachof the orf2 and orf2+3 NheI/SalI fragments was ligated into thePBLUEBAC4.5 NheI/SalI insect cell expression vector (Invitrogen),creating the plasmids PBLUEBAC4.5.NV.orf2 #2 and PBLUEBAC4.5.NV.orf2+3#12 (FIG. 11).

The sequences encoding NV.orf2 or orf2+3 were recombined into theAutographa californica baculovirus (AcNPV) via the PBLUEBAC4.5 transfervector by co-transfecting 2 μg of transfer vector with 0.5 μg oflinearized, wild-type viral DNA into SF9 cells as described (Kitts etal., 1991). Recombinant baculovirus was isolated by plaque purification(Smith et al, 1983). Suspension cultures of 1.5×10⁶ SF9 cells per mlwere harvested following 48 hours of infection with the relevantbaculovirus at a multiplicity of infection (moi) of 2-10 in serum freemedium (Maiorella et al., 1988). The recombinant proteins were detectedin the media using the commercially available RIDASCREEN Norovirusimmunoassay (SciMedx Corporation) (FIG. 12). VLPs were purified from themedia by sucrose gradient sedimentation (see, e.g., Kirnbauer et al. J.Virol. (1993) 67:6929-6936), and the presence of VLPs in peak fractionswas confirmed by electron microscopy (FIG. 13).

Example 3 Production of a Multiepitope Fusion Protein

A polynucleotide encoding an Nterm-NTPase fusion, comprisingapproximately amino acids 1 to 696, numbered relative to NorovirusMD145-12 (SEQ ID NO:13), is isolated from a Norovirus. This construct isfused with a polynucleotide encoding a polymerase polypeptide whichincludes approximately amino acids 1190-1699 of the polyprotein numberedrelative to Norovirus MD145-12. The polymerase-encoding polynucleotidesequence is fused downstream from the Nterm-NTPase-encoding portion ofthe construct such that the resulting fusion protein includes thepolymerase polypeptide at its C-terminus. The construct is cloned intoplasmid, vaccinia virus, adenovirus, alphavirus, and yeast vectors.Additionally, the construct is inserted into a recombinant expressionvector and used to transform host cells to produce the Nterm-NTPase-Polfusion protein.

Example 4 Activation of CD8⁺ T Cells

⁵¹ Cr Release Assay. A ⁵¹Cr release assay is used to measure the abilityof T cells to lyse target cells displaying a Norovirus or Sapovirusepitope. Spleen cells are pooled from the immunized animals. These cellsare stimulated in vitro for 6 days with a CTL epitopic peptide, derivedfrom a Norovirus or Sapovirus, in the presence of IL-2. The spleen cellsare then assayed for cytotoxic activity in a standard ⁵¹Cr release assayagainst peptide-sensitized target cells (L929) expressing class I, butnot class II MHC molecules, as described in Weiss (1980) J. Biol. Chem.255:9912-9917. Ratios of effector (T cells) to target (B cells) of 60:1,20:1, and 7:1 are tested. Percent specific lysis is calculated for eacheffector to target ratio.

Example 5 Activation of Norovirus and Sapovirus-Specific CD8⁺ T Cellswhich Express IFN-γ

Intracellular Staining for Interferon-gamma (IFN-γ). Intracellularstaining for IFN-γ is used to identify the CD8⁺ T cells that secreteIFN-γ after in vitro stimulation with a Norovirus and/or Sapovirusantigen. Spleen cells of individual immunized animals are restimulatedin vitro either with an immunogenic composition described herein or witha non-specific peptide for 6-12 hours in the presence of IL-2 andmonensin. The cells are then stained for surface CD8 and forintracellular IFN-γ and analyzed by flow cytometry. The percent of CD8⁺T cells which are also positive for IFN-γ is then calculated.

Example 6 Proliferation of Norovirus and Sapovirus-Specific CD4⁺ T Cells

Lymphoproliferation assay. Spleen cells from pooled immunized animalsare depleted of CD8⁺ T cells using magnetic beads and are cultured intriplicate with either an immunogenic composition described herein, orin medium alone. After 72 hours, cells are pulsed with 1 μCi per well of³H-thymidine and harvested 6-8 hours later. Incorporation ofradioactivity is measured after harvesting. The mean cpm is calculated.

Example 7 Ability of VP1-VP2 Encoding DNA Vaccine Formulations to PrimeCTLs

Animals are immunized with 10-250 μg of plasmid DNA encoding VP1 and VP2as described in Example 1 and plasmid DNA encoding the Nterm-NTPase-Polfusion protein as described in Example 3. DNA is delivered either byusing PLG-linked DNA (see below), or by electroporation (see, e.g.,International Publication No. WO/0045823 for this delivery technique).The immunizations are followed by a booster injection 6 weeks later ofplasmid DNA encoding Nterm-NTPase-Pol and plasmid DNA encoding VP1 andVP2.

PLG-delivered DNA. The polylactide-co-glycolide (PLG) polymers areobtained from Boehringer Ingelheim, U.S.A. The PLG polymer is RG505,which has a copolymer ratio of 50/50 and a molecular weight of 65 kDa(manufacturers data). Cationic microparticles with adsorbed DNA areprepared using a modified solvent evaporation process, essentially asdescribed in Singh et al., Proc. Natl. Acad. Sci. USA (2000) 97:811-816.Briefly, the microparticles are prepared by emulsifying 10 ml of a 5%w/v polymer solution in methylene chloride with 1 ml of PBS at highspeed using an IKA homogenizer. The primary emulsion is then added to 50ml of distilled water containing cetyl trimethyl ammonium bromide (CTAB)(0.5% w/v). This results in the formation of a w/o/w emulsion which isstirred at 6000 rpm for 12 hours at room temperature, allowing themethylene chloride to evaporate. The resulting microparticles are washedtwice in distilled water by centrifugation at 10,000 g and freeze dried.Following preparation, washing and collection, DNA is adsorbed onto themicroparticles by incubating 100 mg of cationic microparticles in a 1mg/ml solution of DNA at 4 C for 6 hours. The microparticles are thenseparated by centrifugation, the pellet washed with TE buffer and themicroparticles are freeze dried.

CTL activity and IFN-γ expression is measured by ⁵¹Cr release assay orintracellular staining as described in the examples above.

Example 8 Immunization Routes and Replicon Particles SINCR (DC+)Encoding for VP1 and VP2

Alphavirus replicon particles, for example, SINCR (DC+) are prepared asdescribed in Polo et al., Proc. Natl. Acad. Sci. USA (1999)96:4598-4603. Animals are injected with 5×10⁶ IU SINCR (DC+) repliconparticles encoding Norovirus VP1 and VP2 intramuscularly (IM), orsubcutaneously (S/C) at the base of the tail (BoT) and foot pad (FP), orwith a combination of ⅔ of the DNA delivered via IM administration and ⅓via a BoT route. The immunizations are followed by a booster injectionof vaccinia virus encoding VP1. IFN-γ expression is measured byintracellular staining as described in Example 5.

Example 9 Alphavirus Replicon Priming, Followed by Various BoostingRegimes

Alphavirus replicon particles, for example, SINCR (DC+) are prepared asdescribed in Polo et al., Proc. Natl. Acad. Sci. USA (1999)96:4598-4603. Animals are primed with SINCR (DC+), 1.5×10⁶ IU repliconparticles encoding Norovirus VP1 and VP2, by intramuscular injectioninto the tibialis anterior, followed by a booster of either 10-100 μg ofplasmid DNA encoding for VP1, 10¹⁰ adenovirus particles encoding VP1 andVP2, 1.5×10⁶ IU SINCR (DC+) replicon particles encoding VP1 and VP2, or10⁷ pfu vaccinia virus encoding VP1 at 6 weeks. IFN-γ expression ismeasured by intracellular staining as described in Example 5.

Example 10 Alphaviruses Expressing VP1 and VP2

Alphavirus replicon particles, for example, SINCR (DC+) and SINCR (LP)are prepared as described in Polo et al., Proc. Natl. Acad. Sci. USA(1999) 96:4598-4603. Animals are immunized with 1×10² to 1×10⁶ IU SINCR(DC+) replicons encoding VP1 and VP2 via a combination of deliveryroutes (⅔ IM and ⅓ S/C) as well as by S/C alone, or with 1×10² to 1×10⁶IU SINCR (LP) replicon particles encoding VP1 and VP2 via a combinationof delivery routes (⅔ IM and ⅓ S/C) as well as by S/C alone. Theimmunizations are followed by a booster injection of 10⁷ pfu vacciniavirus encoding VP1 at 6 weeks. IFN-γ expression is measured byintracellular staining as described in Example 5.

Example 11 Immunization with Combinations of Norovirus Antigens andAdjuvants

The following example illustrates immunization with various combinationsof NV, SMV and HV antigens in a mouse model. The NV, SMV and HV antigensare prepared and characterized as described herein. CD1 mice are dividedinto nine groups and immunized as follows:

TABLE 3 Immunization Schedule Group Immunizing Composition Route ofDelivery 1 Mixture of NV, SMV, HV Intra-peritoneal or intra-nasalantigens (5 μg/each) + or mucosal (oral) following by CFA parenteral(intra-muscular admin) 2 Mixture of NV, SMV, HV Intra-peritoneal orintra-nasal antigens (5 μg/each) + or mucosal (oral) following by AlOH(200 μg) parenteral (intra-muscular admin) 3 Mixture of NV, SMV, HVIntra-peritoneal or intra-nasal antigens (5 μg/each) + or mucosal (oral)following by CpG (10 ug) parenteral (intra-muscular admin) 4 Mixture ofNV, SMV, HV Intra-peritoneal or intra-nasal antigens (5 μg/each) + ormucosal (oral) following by AlOH (200 μg) + parenteral (intra-muscularadmin) CpG (10 μg) 5 Complete Freunds Intra-peritoneal or intra-nasalAdjuvant (CFA) or mucosal (oral) following by parenteral (intra-muscularadmin) 6 Mixture of NV, SMV, HV Intra-peritoneal or Intranasal (5μg/each) + or mucosal (oral) following by LTK63 (5 μg) parenteral(intra-muscular admin) 7 AlOH (200 μg) + Intra-peritoneal or intra-nasalCpG (10 μg) or mucosal (oral) following by parenteral (intra-muscularadmin) 8 CpG (10 μg) Intra-peritoneal or intra-nasal or mucosal (oral)following by parenteral (intra-muscular admin) 9 LTK63 (5 μg)Intra-peritoneal or intra-nasal or mucosal (oral) following byparenteral (intra-muscular admin)

Mice are immunized at two week intervals. Two weeks after the lastimmunization, all mice are challenged with the appropriate strain. Whenmucosal immunization (e.g., intra-nasal(in)) is used, the animal modelis also challenged mucosally to test the protective effect of themucosal immunogen.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed methods and system of the invention will be apparent to thoseskilled in the art without departing from the scope and spirit of theinvention. Although the invention has been described in connection withspecific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be covered by thepresent invention.

The invention claimed is:
 1. A method of eliciting an immunologicalresponse in a subject, comprising administering to said subject a firstimmunogenic composition comprising: (a) a recombinant polynucleotide,wherein the polynucleotide comprises the nucleotide sequence of SEQ IDNO:2; and (b) a pharmaceutically acceptable excipient.
 2. The method ofclaim 1, wherein the composition is administered parenterally, ormucosally.
 3. The method of claim 2, comprising the following steps: (a)mucosally administering said first immunogenic composition; and (b)parenterally administering a second immunogenic composition comprisingone or more Norovirus antigens.
 4. The method of claim 3, wherein thefirst immunogenic composition and the second immunogenic composition aredifferent.
 5. The method of claim 3, wherein at least one step isperformed two or more times.
 6. The method of claim 3, wherein themucosal administration is intranasal, oral, intrarectal, orintravaginal.
 7. The method of claim 3, wherein the parenteraladministration is transcutaneous.
 8. The method of claim 1, wherein thecomposition further comprises at least one polypeptide from a Norovirus.9. The method of claim 1, wherein the composition further comprises apolynucleotide comprising an ORF1 sequence from a Norovirus.
 10. Themethod of claim 1, wherein the composition further comprises apolynucleotide comprising an ORF2 sequence from a Norovirus.
 11. Themethod of claim 1, wherein the composition further comprises apolynucleotide comprising an ORF3 sequence from a Norovirus.
 12. Themethod of claim 1, wherein the composition further comprises avirus-like particle (VLP).
 13. The method of claim 12, wherein thevirus-like particle is from a Norovirus.
 14. The method of claim 13,wherein the virus-like particle (VLP) comprises at least two antigensfrom different strains of Norovirus.
 15. The method of claim 14, whereinat least one antigen is from a virus selected from the group consistingof a Norwalk virus (NV), a Snow Mountain virus (SMV), an Hawaii virus(HV) and combinations thereof.
 16. The method of claim 1, wherein thecomposition further comprises a microparticle.
 17. The method of claim16, wherein said microparticle is a poly(L-lactide), poly(D,L-lactide)or poly(D,L-lactide-co-glycolide) microparticle.